Tracing the evolution of key traits in dorid nudibranchs

Silvia Prieto-Baños; Kara K S Layton

doi:10.1371/journal.pone.0317704

. 2025 Apr 2;20(4):e0317704. doi: 10.1371/journal.pone.0317704

Tracing the evolution of key traits in dorid nudibranchs

Silvia Prieto-Baños ^1,^2,³, Kara K S Layton ^1,^4,^*

Editor: Satheesh Sathianeson⁵

PMCID: PMC11964261 PMID: 40173132

Abstract

Reconstructing trait evolution is critically important for elucidating the processes generating biodiversity. However, this work is in its infancy in non-model clades for which we lack a basic understanding of their ecology and biology. Here, we compile information about prey preference, chemical acquisition and colour pattern in dorid nudibranchs (Nudibranchia: Doridoidei) and reconstruct their ancestral states using a multi-gene phylogeny to investigate the evolution of these key traits. Our analyses show that the most recent common ancestor (MRCA) of Doridoidei preferred sponge prey from which they sequestered metabolites, and subsequent shifts to different prey types and de novo synthesis of defensive compounds occurred multiple times independently across the phylogeny. Additionally, the MRCA likely exhibited complex colour patterns, including spots or stripes, with uniform morphotypes evolving in most families. Despite the fact that many dorid nudibranchs derive both metabolites and pigments from their prey, we found no evidence of correlated evolution amongst these traits. As part of this work, we present a multi-gene phylogeny for Doridoidei with representatives from 88 genera and 18 families, but there remain issues with poor support across the tree. Nonetheless, for the first time, we explore the evolution of key traits that contributed to the diversification of dorid nudibranchs, highlighting the need for more refined trait data and greater phylogenetic resolution for future work.

1. Introduction

Understanding the ecological factors underpinning speciation and the generation of biodiversity is of utmost importance in evolutionary biology. Despite this, we lack the fundamental ecological knowledge required for this work, and this is especially true for understudied invertebrate groups. Nudibranch molluscs (Gastropoda: Heterobranchia) exhibit remarkable diversity on multiple scales and serve as unique and interesting systems in ecology and evolutionary biology. The Doridoidei infraorder (herein referred to as dorids) is one of the most speciose nudibranch groups, comprising over 2,000 species [1–3]. Dorids are globally distributed, occupying a wide range of marine environments and ecological niches, and they exhibit diverse behavioural and morphological traits, including dietary specialization [4,5], aposematic colour patterns [6,7] and chemical defense [8,9]. Nudibranchs feed largely on other invertebrates [3] but their feeding preferences, and degree of specialization, likely vary across taxonomic groups and ecological contexts. For instance, Bathydoridoidei, the comparably species poor sister group to Doridoidei, are generalists that feed on multiple marine invertebrate phyla (e.g., echinoderms, bryozoans, crustaceans) [10], while most members of the Doridoidei are thought to have more specialized diets [11]. Broadly speaking, dietary specialization and prey shifts have been invoked as drivers of diversification in marine heterobranchs [5,12], but how chemical defense and colour pattern variation might interact and play a role is largely unknown.

Nudibranchs are known to house diverse chemical compounds that play a key role in predator avoidance [11]. In fact, the bioactive metabolites isolated from nudibranchs have received considerable attention from natural products researchers for their potential use in antiviral and anticancer pharmaceuticals (e.g., [13]). The diverse chemical compounds found in nudibranchs are typically sequestered from their prey or de novo synthesized. In the case of sequestration, bioactive compounds can be selectively stored while others are discarded [14], and in some cases inactive compounds may be secondarily modified [13]. For instance, Chromodoris nudibranchs selectively sequester bioactive latrunculin A in specialized mantle glands [14], while Felimida secondarily modifies sesquiterpene from their sponge prey [15]. Conversely, Dendrodoris grandiflora has been shown to de novo synthesize the sesquiterpene polygodial [16]. Previous work has characterized chemical diversity and the mode of chemical acquisition in some nudibranchs (e.g., [13,14,17]) but whether this is phylogenetically conserved remains unknown.

Chemically-defended species often exhibit bright colours to advertise their toxicity or unpalatability to predators in a strategy known as aposematism [18]. Nudibranchs display a fascinating diversity of colour patterns and serve as ideal models for understanding aposematism in marine systems. Recent work has shown considerable variation in both chemical defence and warning signals (colour patterns) amongst aposematic nudibranch species, especially those involved in Müllerian mimicry rings- where multiple, toxic species share similar colour patterns (e.g., [9]). Several other studies have shown that colour patterns are unreliable as diagnostic morphological characters since distantly-related species can share similar colour patterns and conversely colour patterns can vary drastically within species [7,19,20]. Despite these studies, our understanding of the evolution of colour patterns in nudibranchs is truly in its infancy, especially compared to terrestrial species (e.g., Heliconius, [21]). Across diverse terrestrial taxa, aposematic species tend to display high-contrast spots or bands while cryptic (camouflaged) species tend to exhibit irregular blotches or stippling, and stripes can be used in both aposematism or crypsis depending on environmental complexity [22–24]. These same colour pattern elements appear across diverse nudibranch taxa but whether they serve the same function in marine species is unknown. Prey preference, chemical acquisition and colour pattern have been described and studied across several nudibranch groups (see above), but no study has investigated the evolution of these traits in tandem, especially in a phylogenetic context.

Key to understanding trait evolution is the presence of a robust phylogeny for which to track trait gain and loss over evolutionary time. Here, we reconstruct Doridoidei phylogeny using publicly available sequence data and we employ ancestral state reconstruction to understand how prey preference, chemical acquisition and colour pattern have evolved over time in dorids. In doing so, we discuss major phylogenetic relationships within Doridoidei, we identify the most likely character states of each ancestral node in the tree, and we look for evidence of correlated evolution amongst traits. For the first time, this study investigates the role that ecological traits have played in shaping dorid nudibranch biodiversity and evolution and in doing so, compiles one of the most extensive ecological datasets for dorid nudibranchs to date.

2. Methods

Compiling genetic and trait data

Sequence data and trait data were compiled for 88 genera and 18 families of dorid nudibranch from publicly available resources (Table 1; Fig 1). One representative species per genus was used for all analyses since, in most cases, trait data was only available at this taxonomic rank. The representative taxa chosen for this study were those that had sufficient genetic data to support phylogenetic reconstruction (e.g., data for at least two of five genes where possible). Five molecular markers were employed for phylogenetic inference and ancestral state reconstruction (ASR), including two mitochondrial (COI, 16S) and three nuclear genes (28S, 18S, H3). All molecular data was mined from GenBank for this work and corresponding accession numbers are available in Table 1. Trait data was obtained from research articles and citizen science databases provided in S1 File. Trait data is resolved at the genus-level because it was lacking for many species, but where it was also lacking at the genus-level we either marked this as missing data or we assigned traits based on confamilial data where there was little variation at the family-level (see Table 1). For each dorid genus, its preferred prey was categorized at the phylum-level, except for one genus where prey preference was characterized as ‘generalist’ because records indicated multiple possible prey sources. Chemical acquisition was categorized as sequestration or de novo synthesis, but this information was missing for several taxa. Where secondary modification of a chemical obtained from prey was reported in the literature, we considered these as sequestration. Lastly, colour patterns were categorized as uniform, with spots or stripes, mottled or with a distinct mantle rim. The latter was included here because the distinct, coloured mantle rim (e.g., Chromodoris, Fig 1I) may be an important anti-predator visual signal in nudibranchs [6]. Conversely, the mottled colour pattern refers to large, undefined blotches or stippling on the mantle (e.g., Aphelodoris, Fig 1R) compared to defined spots (e.g., Dendrodoris, Fig 1B). Where tubercles or papilla on the mantle had distinctly coloured tips (e.g., Cadlinella, Fig 1F), these were considered visually similar to spots and thus the colour pattern was defined as such. All colour pattern data was retrieved from specimen images on the Sea Slug Forum and iNaturalist. All taxonomic names were verified on the World Register of Marine Species (WoRMS).

Table 1. Sequence data and trait data for all dorid taxa in this study. GenBank accession numbers are provided for each gene. Prey type is characterized at the phylum-level. Chemical acquisition is characterized as sequestration or de novo synthesis. Colour pattern is characterized as uniform, with stripes or spots, mottled or with a distinct mantle rim. References are available in S1 File. Missing data is represented by a dash. Data marked with an asterisk indicates that genus-level information was lacking and trait data was instead assigned based on family-level patterns or supporting data, provided in parentheses.

Species	GenBank Accessions					Prey Group	Chemical Acquisition	Colour Pattern
	COI	16S	28S	18S	H3
Actinocyclidae
Actinocyclus verrucosus	MF958438	MF958311	MF958397	MF958352	–	Porifera ¹	–	Mottled
Hallaxa translucens	EU982760	EU982814	KT698821	MF958341	–	Porifera & Chordata^2-4	–	Uniform
Aegiridae
Aegires flores	MF958442	MF958316	MF958402	–	–	Porifera^2,3,5-8	Sequestration ⁹	Stripes/spots & distinct rim
Notodoris minor	KP871631	KP871678	–	–	KP871654	Porifera ^10,11	Sequestration ^12,13	Stripes/spots
Akiodorididae
Akiodoris lutescens	–	MN224076	MN224112	–	–	Bryozoa ¹⁴	–	Uniform
Armodoris anudeorum	KP340387	KP340290	KP340355	–	KP340412	–	–	Uniform
Cadlinellidae
Cadlinella ornatissima	MF958415	MF958284	MF958371	MF958325	–	Porifera ²	Sequestration* ¹⁵ (presence of MDFs)	Stripes/spots
Cadlinidae
Aldisa sanguinea	MF958435	MF958309	MF958394	MF958350	–	Porifera ¹⁰	Sequestration ¹⁰	Uniform
Cadlina laevis	MN224049	MN224081	MN224116	–	–	Porifera ^{8, 10, 16-18}	Sequestration ^{10, 19} & synthesis ¹⁰	Stripes/spots & distinct rim
Inuda luarna	EU982718	EU982768	–	–	–	Porifera* (confamilial trait)	Sequestration* ²⁰ (presence of MDFs)	Uniform
Calycidorididae
Calycidoris guentheri	KP340397	KP340301	KP340371	KP340338	KP340417	Bryozoa* (confamilial trait)	Synthesis* (absence of mantle glands; confamilial trait)	–
Diaphorodoris lirulatocauda	KP340403	KP340307	KP340377	KP340344	KP340422	Bryozoa ^{2, 8, 21}	Synthesis ²²	Stripes/spots & distinct rim
Chromodorididae
Ardeadoris scottjohnsoni	EU982714	EU982763	KT698766	–	–	Porifera ²³	Sequestration ²⁴	Stripes/spots & distinct rim
Ceratosoma brevicaudatum	–	EU512141	EU512052	–	–	Porifera ^{25, 26}	Sequestration ^{25, 26}	Stripes/spots & distinct rim
Chromodoris magnifica	EF535110	EF534042	EF534028	–	–	Porifera ^{27, 28}	Sequestration ²⁸	Stripes/spots & distinct rim
Chromolaichma edmundsi	HM162686	HM162595	MF958390	MF958347	HM162595	Porifera ²⁹	Sequestration ³⁰	Distinct rim
Diversidoris aurantinodulosa	EF535141	EF534069	–	EF534011	–	Porifera ⁵	Sequestration* (confamilial trait)	Stripes/spots & distinct rim
Doriprismatica stellata	KT600693	KT595622	KT698782	–	–	Porifera ³¹	Sequestration ³¹	Mottled & distinct rim
Felimare tema	HM162685	HM162594	MF958389	MF958346	HM162594	Porifera ³²	Sequestration ^{10, 33}	Stripes/spots & distinct rim
Glossodoris buko	KT600711	KT595638	KT698808	–	–	Porifera ²⁵	Sequestration ^{25, 29}	Stripes/spots, mottled & distinct rim
Goniobranchus reticulatus	JQ727853	JQ727733	–	–	–	Porifera ^{24, 25}	Sequestration ^{24, 25}	Stripes/spots, mottled & distinct rim
Hypselodoris obscura	MG645598	MG645438	–	–	MG645519	Porifera ¹⁰	Sequestration ¹⁰	Stripes/spots, mottled & distinct rim
Mexichromis antonii	EU982748	EU982800	–	–	MG645548	Porifera ²	Sequestration* (confamilial trait)	Stripes/spots & distinct rim
Miamira striata	MW892629		–	–	MW883960	Porifera ¹⁰	Sequestration ¹⁰	Stripes/spots, mottled & distinct rim
Thorunna florens	JQ727913	JQ727817	–	–	–	Porifera ³	Sequestration* (confamilial trait)	Stripes/spots & distinct rim
Tyrinna evelinae	EU982757	EU982811	MF958391	–	–	Porifera ^{18, 34}	Sequestration ¹⁰	Stripes/spots
Verconia verconis	EF535118	EF534046	–	EF534036	–	Porifera ²	Sequestration* (confamilial trait)	Stripes/spots, mottled & distinct rim
Corambidae
Corambe obscura	KP340399	KP340303	KP340373	KP340340	KP340419	Bryozoa ^{2, 35}	–	Uniform
Dendrodorididae
Dendrodoris densoni	–	MF958308	MF958393	MF958349	–	Porifera ^{2, 36}	Synthesis ^{25, 36, 37}	Stripes/spots & mottled
Doriopsilla janaina	–	MF958312	MF958398	MF958353	–	Porifera ^{2, 38-40}	Synthesis ^{10, 40}	Stripes/spots
Discodorididae
Asteronotus cespitosus	MF958419	MF958288	MF958375	MF958328	MN720324	Porifera ^{9, 41}	Sequestration ^{9, 41}	Uniform
Atagema kimberlyae	OQ362152	–	–	–	OQ366206	Porifera ²	–	Stripes/spots
Carminodoris flammea	MN720285	–	–	–	MN720311	–	–	Mottled
Diaulula sandiegensis	KP871647	KP871695	–	–	–	Porifera ^{2, 10}	Sequestration ^{10, 42}	Stripes/spots
Discodoris coerulescens	MF958421	MF958290	MF958377	MF958330	–	Porifera ²	Sequestration ⁴³	Mottled
Geitodoris heathi	KP871642	KP871690	–	–	KP871666	Porifera ²	–	Mottled
Halgerda dalanghita	MF958420	MF958289	MF958376	MF958329	MN720316	Porifera ⁹	Sequestration ^{9, 25}	Stripes/spots, mottled & distinct rim
Hoplodoris nodulosa	FJ917486	FJ917428	FJ917469	FJ917443	–	–	–	Mottled
Jorunna tomentosa	AJ223267	AJ225191	–	–	–	Porifera ⁴⁴	Sequestration ^25,45	Stripes/spots
Paradoris liturata	KP871648	KP871696	–	–	–	Porifera ⁴³	–	Stripes/spots
Peltodoris nobilis	HM162684	HM162593	–	–	HM162499	Porifera ^{10, 46-48}	Sequestration ^{10, 48}	Mottled
Platydoris sanguinea	MF958416	MF958285	MF958372	MF958326	MN720312	Porifera ⁴⁹	Synthesis ⁴⁰	Mottled
Rostanga pulchra	GQ292028	–	–	GQ326864	–	Porifera ²	–	Uniform
Sclerodoris tuberculata	MF958417	MF958286	MF958373	MF958327	MN720323	Porifera ²	Synthesis ¹⁰	Uniform
Taringa telopia	MN720291	KP871700	–	–	KP871675	Porifera* ⁵⁰ (confamilial trait)	–	Stripes/spots, mottled & distinct rim
Thordisa albomacula	MF958418	MF958287	MF958374	–	–	Porifera ²	–	Uniform
Dorididae
Aphelodoris sp.	MF958424	MF958293	MF958379	MF958332	–	Porifera ^{2, 51}	–	Mottled
Conualevia alba	KC153021	KC153023	–	–	–	Porifera ⁵²	–	Uniform
Doriopsis granulosa	AF249798	AF249223	–	AF249212	–	Porifera ²	–	Uniform
Doris pseudoargus	AJ223256	AJ225180	–	–	–	Porifera ⁵³	Synthesis ^54-57	Mottled
Goniodorididae
Ancula gibbosa	KP340388	KP340291	KP340356	KP340322	KP340413	Entoprocta ^{2, 8, 58-61}	–	Stripes/spots
Goniodoridella savignyi	OK143202	–	–	–	–	Bryozoa ³ & Chordata¹¹⁰	–	Stripes/spots, mottled & distinct rim
Goniodoris nodosa	AF249788	AF249226	–	AJ224783	–	Bryozoa¹¹¹ & Chordata ^{53, 62}	Sequestration^63,64	Mottled & distinct mantle rim
Lophodoris danielsseni	OK156412	–	–	–	OK169877	Bryozoa¹¹² Chordata (confamilial trait) & Entoprocta ²	–	Uniform
Murphydoris puncticulata	OK156427	–	–	–	OK169891	Bryozoa ² & Chordata¹¹²	–	Stripes/spots, mottled & distinct rim
Okenia vena	KY661381	KY661373	–	–	KY661384	Bryozoa ³	Sequestration ^{10, 65}	Mottled & distinct rim
Trapania reticulata	MF958432	MF95803	–	MF958342		Bryozoa ³ & Entoprocta ^{3, 66, 67}	–	Stripes/spots & mottled
Hexabranchidae
Hexabranchus sanguineus	MF958433	MF958305	MF958388	MF958344	–	Porifera ¹⁰	Sequestration ^{10, 25}	Mottled & distinct rim
Mandeliidae
Mandelia mirocornata	MF958411	MF958278	MF958365	MF958321	–	Porifera ⁶⁸ (based on buccal morphology of sponge-feeding dorids)	–	Stripes/spots
Onchidorididae
Acanthodoris nanaimoensis	KM219657	KJ653656	KP340360	KP340325	KM225810	Bryozoa ^{2, 70}	Synthesis ^{10, 71}	Distinct rim
Adalaria slavi	MN224050	MN224074	MN224110	MN224105	–	Bryozoa ^{2, 8, 10, 72, 73}	Sequestration ¹⁰	Uniform
Atalodoris oblonga	KP340410	–	KP340385	KP340349	KP340430	Bryozoa ^{2, 74}	–	Mottled
Onchidoris muricata	KM219680	–	KP340383	KP340348	KM225830	Bryozoa ^75-78	–	Mottled
Onchimira cavifera	MN224073	MN224104	MN224137	MN224109	–	Bryozoa ⁷³	–	Uniform
Phyllidiidae
Ceratophyllidia sp.	MF958413	MF958281	MF958368	MF958323	–	Porifera* (confamilial trait)	Sequestration* (confamilial trait)	Stripes/spots
Phyllidia coelestis	MF958412	MF958279	MF958366	–	–	Porifera ^{9, 79-81}	Sequestration ^{9, 79-83}	Stripes/spots
Phyllidiella nigra	–	MF958280	MF958367	MF958322	–	Porifera ⁸⁴	Sequestration ^85-87	Stripes/spots
Phyllidiopsis krempfi	KX235972	–	–	–	–	Porifera* (based on buccal morphology of sponge-feeding phyllids)	Sequestration ¹⁰	Stripes/spots
Reticulidia halgerda	MF958414	MF958282	MF958369	–	–	Porifera ^{2, 88}	Sequestration ¹⁰	Stripes/spots
Polyceridae
Colga pacifica	MZ782097	–	–	–	–	–	–	Uniform
Crimora lutea	EF142903	EF142950	–	–	–	Bryozoa ^{2, 88}	–	Stripes/spots
Gymnodoris ceylonica	KY806818	KY806790	KY806809	KY806800	–	Mollusca ^89-91	–	Stripes/spots & distinct rim
Kalinga ornata	MN224072	MN224103	MN224136	–	–	Generalist ^92-94	–	Stripes/spots
Kaloplocamus sp.	MF958429	MF958299	MF958383	MF958337	–	Bryozoa ^{2, 5}	–	Mottled
Lecithophorus capensis	MZ382782	–	–	–	MZ399572	Bryozoa ^{2, 95}	–	Uniform
Limacia sp.	HM162692	HM162602	KP340353	KP340320	HM162508	Bryozoa ^{96, 97}	Synthesis ⁹⁸	Stripes/spots
Martadoris mediterranea	KP793057	–	–	–	KP793060	–	–	Mottled
Nembrotha cristata	MF958431	MF958301	MF958385	MF958339		Chordata ^{10, 25, 99}	Sequestration ^{10, 25, 100}	Stripes/spots & mottled
Palio dubia	AJ223272	AJ225197	–	–	–	Bryozoa ¹⁰¹	–	Mottled
Plocamopherus pecoso	MF958430	MF958300	MF958384	MF958338	–	Bryozoa ^{5, 40}	Synthesis ¹⁰²	Mottled
Polycera aurantiomarginata	JX274068	JX274038	–	–	–	Bryozoa ^{2, 103, 104}	Sequestration ^{204, 205} & synthesis* (some Polycera species contain a chemical that is biosynthesized in other nudibranchs) _{10, 102}	Stripes/spots & distinct rim
Roboastra gracilis	EF142863	EF142912	–	–	–	Bryozoa ^{2, 106, 107}	Sequestration ^{10, 106}	Stripes/spots
Tambja marbellensis	HM162689	HM162599	–	–	HM162505	Bryozoa ^{2, 10, 107}	Sequestration ^{10, 107, 108}	Stripes/spots & distinct rim
Thecacera sp.	MZ382795	–	–	–	MZ399586	Bryozoa ²	Synthesis ¹⁰²	Stripes/spots
Triopha catalinae	HM162690	HM162600	KP340354	KP340321	HM162506	Bryozoa ^{2, 10, 98, 109}	Sequestration & synthesis^{10, 98}	Stripes/spots
Tyrannodoris ernsti	KJ999212	–	–	–	KJ999232	Mollusca ^{5, 105}	Sequestration ¹⁰	Stripes/spots
Vayssierea sp.	MZ382796	–	MF958408	MF958362	MZ399587	Annelida ^{2, 3, 69}	–	Uniform
Showajidaiidae
Showajidaia sagamiensis	MN224070	MN224101	MN224134	MN224108	–	–	–	Stripes/spots
Outgroup
Bathydoris aioca	KP871635	KP871682	–	–	KP871658

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Fig 1 — (A) Cadlinidae: *Aldisa* (R. Agarwal), (B) Dendrodorididae: *Dendrodoris* (J. Brodie), (C) Mandeliidae: *Mandelia* (Seascapeza), (D) Phyllidiidae: *Phyllidiopsis* (B. Picton), (E) Actinocyclidae: *Actinocyclus* (B. Rudman), (F) Cadlinellidae: *Cadlinella* (K. Layton), (G) Hexabranchidae: *Hexabranchus* (C. Watanabe), (H) Showajidaiidae: *Showajidaia* (I. Diver), (I) Chromodorididae: *Chromodoris* (K. Layton), (J) Polyceridae: *Polycera* (S. Verheyen), (K) Polyceridae: Okadaiinae: *Vayssierea* (I. Diver), (L) Calycidorididae: *Diaphorodoris* (J. Yasaki), (M) Dorididae: *Doris* (B. Picton), (N) Corambidae: *Corambe* (R Agarwal), (O) Onchidorididae: *Onchidoris* (B. Picton), (P) Goniodorididae: *Goniodoris* (B. Picton), (Q) Aegiridae: *Aegires* (G. Cobb), and (R) Discodorididae: *Discodoris* (P. Bourjon).

Phylogenetic and ancestral state reconstruction

Sequence data for all five markers was downloaded from GenBank and aligned with MAFFT v7.52 [25] before concatenation in Geneious v.9 (https://www.geneious.com). A maximum-likelihood tree was obtained for this concatenated dataset using IQ-TREEv2.2.0 [26] with a GTR + G + I model and 1000 ultrafast bootstrap replicates and with Bathydoris as an outgroup [1]. A Bayesian tree was obtained with MrBayes v3.2 [27] using a GTR + G + I model, 10,000,000 MCMC generations, a 25% burnin and four Markov chains, with trees sampled every 100 generations. As described above, we conducted an extensive literature search to generate trait data for contemporary taxa as input for ASR. The final ML tree was used alongside this trait data for ASR in the corHMM package in R [28]. This package, and the rayDISC function specifically, was chosen for ASR since it accepts both missing data and polymorphic character states (e.g., members of some genera can both synthesize and sequester defensive compounds; members of some genera exhibit spots/stripes while others are uniform). This functionality was critically important since nudibranch traits are complex and, in some cases, unknown. We conducted ASR with both Equal Rates (ER) and All-Rates-Different (ARD) models and employed the model that had the lowest corrected Akaike Information Criterion (AICc) score. In all cases, the ER model was the best fit and was executed with default parameters and marginal likelihoods in corHMM. Lastly, we tested for correlated evolution among our three traits using an independent contrasts correlation model in an MCMC framework via a two-step process in BayesTraits v4.1.1 [29]. First, we ran the ‘complex’ analysis, using a sample period of 1,000 with 1,010,000 iterations and a burn-in of 10,000, and then a stepping stone sampler of 100 stones each run for 1000 iterations, to calculate the covariance between each pair of traits. Then, we ran the ‘simple’ analysis using the same parameters above but using the TestCorrel command to set the covariance to zero for each pair of traits. Each analysis generated a single log marginal likelihood and these were used to calculate Log Bayes Factors (Log BF) = 2(log marginal likelihood complex model – log marginal likelihood simple model). Log BF values of < 2 are considered weak evidence of correlation [29].

3. Results and Discussion

Is the Doridoidei phylogeny resolved?

Several recent papers have employed multi-marker datasets to reconstruct dorid nudibranch phylogeny with variable results [1,30,31]. Here, we employed IQ-TREE to generate a concatenated ML phylogeny for 88 genera of Doridoidei (Fig 2), with a final alignment length of 3,603 bp. We recovered patterns similar to [1,30] and [31], but with some notable differences. First, we recover Phyllidiidae at the base of the tree with Mandeliidae, but [31] recover Mandeliidae as sister to Aegiridae and [1] do not include Mandeliidae. We also recover a recurring pattern with Dorididae. In our results, Dorididae is polyphyletic because Aphelodoris falls outside this family and instead is sister to the Discodorididae + Aegiridae. This is partially consistent with the results from [30], where Dorididae is also polyphyletic due to Aphelodoris grouping with Discodorididae, although not with Aegiridae. In contrast, [31], who have dense sampling within Discodorididae, recover Dorididae as monophyletic, but their dataset lacks Aphelodoris.

Fig 2 — Non-monophyletic families are marked with an asterisk. Hash marks denote that the branch has been truncated to one half of its original length.

One of the most notable topological differences is the position of Actinocyclidae, which is sister to some, but not all families, in our phylogeny (BS = 84) (i.e., Mandeliidae + Phyllidiidae, Dendrodorididae and Cadlinidae are at the base of the tree), similar to [31]. Conversely, some earlier results recover Actinocyclidae as sister to all other Doridoidei families [1,30]. The position of Discodorididae also varies tremendously, with Discodorididae sister to Aegiridae in our study, albeit with weak support (BS = 74), compared to earlier results that show Discodorididae sister to Dorididae [30] or Goniodorididae [1]. Additionally, the clade containing Cadlinellidae, Hexabranchidae and Showajidaiidae is sister to Chromodorididae in our phylogeny (BS = 87) but sister to Polyceridae in [30], unresolved in [1] and sister to both Chromodorididae and Polyceridae in [31]. We also conducted a Bayesian analysis (S1 Fig) but many deeper nodes (i.e., relationships among families) were poorly supported and subsequently collapsed into polytomies. Despite this poor resolution, we still recover similar family-level relationships as in our ML analysis, except that Polyceridae is non-monophyletic. Another difference between the ML and Bayesian analysis is the position of Goniodorididae which appears as sister to a clade containing Aegiridae and Discodorididae in the former but as sister to the rest of the Doridoidei in the latter. However, the result from the Bayesian analysis has never been recovered in any molecular or morphological phylogenies to date. This significant topological variability among methods and studies likely reflects differences in taxon and gene sampling as well as phylogenetic methodology. For instance, [1] and [30] include 56 genera while here we include all 88 genera for which genetic data is currently available. Additionally, [1,30] and [31] employ three or four gene datasets, spanning a total of five unique genes, for which we incorporate data from all five loci here, however with variable completeness across our dataset. In any case, we report multiple instances of poor support within Doridoidei (i.e., BS < 95) and these relationships should be interpreted with caution until additional genomic data becomes available for phylogenetic reconstruction.

Due to the many examples of poor support across Doridoidei, future work should employ reduced representation or whole genome approaches to generate larger datasets that have greater power for phylogenetic resolution. In fact, transcriptomes [32,33], exons [34], ultraconserved elements (UCEs) [35] and mitogenomes [36] have already been used to resolve both deep and shallow evolutionary relationships in heterobranchs, but these studies did not target Doridoidei specifically. With respect to taxon sampling, we restricted our dataset to just a single representative per genus and while this might contribute to topological uncertainty, the lack of a densely sampled phylogeny across these same genera limits this investigation. One oddity is the long branch leading to Vayssierea, an intertidal nudibranch genus currently residing in the Okadaiinae subfamily within Polyceridae. This pattern could reflect either undersampling of this subfamily or accelerated evolutionary rates, the latter of which has recently been uncovered in chromodorid nudibranchs [37]. Lastly, we employ IQ-TREE for ML analysis while previous studies mostly employed RAxML [38], which may contribute to the topological variation observed here. In all, there remain issues with poor support across the Doridoidei phylogeny that is unlikely to be resolved without additional sampling, both taxonomic and genomic. Even with denser sampling, we may continue to face challenges when reconstructing dorid nudibranch phylogeny.

How did chemical defence, prey preference and colour patterns evolve in Doridoidei?

Ancestral state reconstruction recovered the most recent common ancestor (MRCA) of all Doridoidei as a sponge-feeder with complex colour patterns (e.g., stripes or spots) that sequestered chemicals from its prey (Fig 3a–3c). Looking at chemical acquisition, de novo synthesis has evolved multiple times independently from a sequestering ancestor (Fig 3a). Interestingly, members of Bathydoridoidei (the sister group to Doridoidei) tend to de novo synthesize and thus a switch to a putatively less costly mechanism, like sequestration (e.g., [39]), may also have facilitated the diversification of Doridoidei. Within Doridoidei, both modes of chemical acquisition are reported from sponge and bryozoan feeders. Entoproct feeding taxa are reported as sequesterers in the literature. Little is known about secondary metabolites in this unique prey group and thus the taxa reported here as entoproct feeders may in fact feed on and sequester metabolites from a more diverse suite of prey. We found little variation in chemical acquisition at the genus level, with the exception of Polycera and Cadlina where different species, and even populations of the same species, have been shown to both sequester and synthesize (e.g., [40]). We expected to find that prey switching evolved concurrently with switches to different chemical acquisition modes, but there is little support for this. However, it is important to note that data on chemical acquisition in nudibranchs is patchy and, in some cases, may be speculative. As such, the results presented here should be interpreted with caution until more robust data is available for re-investigation.

Fig 3 — Pie charts at each node represent marginal likelihoods of ancestral states. Polymorphic character states are marked with two or more data points in extant taxa which are labeled with genus names. Taxa without a data point are missing the relevant trait data. Family names are provided at the relevant node and non-monophyletic families are marked with an asterisk. Hash marks denote that the branch has been truncated to one half of its original length. Inset in (c) shows examples of each colour pattern. From left to right, top to bottom, with image attributions in parentheses: *Adalaria* (B. Picton), *Polycera* (S. Verheyen), *Hoplodoris* (S. Rorhlach) and *Ardeadoris* (S. Graham).

Looking at prey preference, feeding on bryozoans most likely evolved four times independently from a sponge feeding ancestor at relatively deep nodes of the phylogeny (Fig 3b). This includes in the MRCA of 1) Polyceridae, 2) Akiodorididae and Calycidorididae, 3) Onchidorididae and Corambidae, and 4) Goniodorididae. However, prey preference is complex in Goniodorididae and thus it is possible that the MRCA fed on entoprocts, bryozoans and other diverse taxa. Feeding on chordates (tunicates) evolved at least three times, from both sponge and bryozoan feeding ancestors. Feeding on molluscs has evolved twice, only within the Polyceridae, from a bryozoan feeding ancestor. Conversely, entoproct feeding may have evolved multiple times within Goniodorididae, or once in the MRCA and then subsequently lost in some taxa, being replaced with bryozoan and tunicate feeding. Alternatively, it is possible that goniodorids are feeding on entoprocts that are found amongst bryozoans and tunicates and in fact this feeding preference may be more prevalent than reported in the literature. Interestingly, sponge feeding was not re-gained in any of the lineages where prey-switching occurred. Most genera feed on specific prey groups, with the exception of most goniodorids that feed on two types of organisms (bryozoans and tunicates or tunicates and entoprocts), the generalist genus Kalinga and the genus Hallaxa (sponges and ascidians). These findings align, in part, with previous work that investigated the evolution of prey preference in cladobranch slugs and uncovered widespread hydrozoan feeding that originated in the MRCA with subsequent switches to specific prey groups [5]. Additionally, Bathydoridoidei, the sister clade to Doroidei, is a much less diverse taxonomic group that tends to exhibit generalist feeding behaviour [10]. As such, the switch to sponge-feeding in the MRCA of Doridoidei may have facilitated their impressive diversification.

Nudibranch colour patterns are complex and their use in signaling may be conditional or vary across different ecological contexts (i.e., what might be considered aposematic in one environment is cryptic in another). Nonetheless, we recover the MRCA of all Doridoidei as having spots or stripes, suggesting that the ancestor displayed complex patterns and that more cryptic patterns subsequently evolved in multiple lineages (Fig 3c). Given the complexity of these colour patterns (i.e., multiple possible colour patterns are present within a single genus) it is also likely that the MRCA(s) exhibited a combination of these patterns, and it is likely that the full spread of colour pattern variation within genera is not captured here. Nonetheless, previous work also recovered a spotted phenotype in the MRCA of a clade of weevils that display diverse colour patterns ranging from uniform to complex net-like patterns [41], but in contrast, complex colour patterns evolved from a uniform ancestor in aposematic beetles [42]. Looking across dorid families, both Chromodorididae and Goniodorididae appear to have the most complex and variable colour patterns, where stripes/spots, mottled patterns and distinct mantle rims appear within many genera. In fact, a distinct mantle rim was most common in Chromodorididae and Goniodorididae, and evolved from spotted/striped, mottled and even uniform ancestors, but it was absent in Phyllidiidae, Akiodorididae, Calycidorididae, Dorididae and Corambidae. Conversely, uniform species were most common in the Akiodorididae, Calycidorididae, Dorididae, Corambidae, and Onchidorididae, and absent from the Chromodorididae. Colour pattern was only consistent in a single family, with all members of the Phyllidiidae exhibiting spots or stripes. The mottled colour pattern occurs multiple times across diverse taxonomic groups so it’s likely that this pattern is conditional in that it ranges from aposematic to cryptic depending on the context. Given the link between diet, chemical defence and colour patterns (e.g., [11,43]), a signal of correlated evolution amongst these three traits might be expected, however, we find weak evidence of this in our BayesTraits analysis (LogBF = 1.73). The lack of evidence for correlated evolution may reflect missing trait data or the lack of fine-scale resolution and thus future work should look to investigate the evolution of these traits across a species-level phylogeny to elucidate clearer patterns.

4. Conclusions

Here, for the first time, we investigate, in tandem, the evolution of prey preference, chemical acquisition and colour pattern in dorid nudibranchs. We use previously published genetic and trait data to support this work, demonstrating the importance of existing databases in advancing our understanding of evolution in non-model, and typically data-deficient, organisms. Despite these advances, there are some key limitations of this work. First, the dorid phylogeny employed for ancestral state reconstruction remains partially unresolved and thus a more comprehensive, species-level phylogeny is needed to confirm the patterns shown here. Additionally, much of the trait data retrieved from the literature is based on just a few representative species per genus or family and thus it is likely that these traits vary both within and among genera and our results might instead reflect broader patterns. Future work should look to revisit these findings and patterns with a fully resolved phylogeny and refined trait data, although more work is needed to improve our understanding of basic biology and ecology in these organisms to support this. Nonetheless, limiting our work to well-sampled and well-known taxonomic groups will only continue to contribute to the pronounced taxonomic bias in evolutionary biology.

Supporting information

S1 Fig. Multi-gene Bayesian phylogeny (COI,16S,18S,28S,H3) for Doridoidei with posterior probabilities provided at each node. Hash marks denote that the branch has been truncated to one half of its original length.

(TIF)

pone.0317704.s001.tif^{(663.9KB, tif)}

S1 File. Supplementary references (Table 1).

(DOCX)

pone.0317704.s002.docx^{(30.5KB, docx)}

Acknowledgments

We thank two anonymous reviewers for their helpful feedback on this manuscript.

Data Availability

Trait data is available within the manuscript and the sequence alignment employed for analysis is available on Dryad: 10.5061/dryad.8sf7m0d0m.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. 2025 Apr 2;20(4):e0317704. doi: 10.1371/journal.pone.0317704.r001

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PLoS One. doi: 10.1371/journal.pone.0317704.r002

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PONE-D-24-20562Tracing the evolution of key traits in dorid nudibranchsPLOS ONE

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the manuscript, the authors traced the evolution of key traits in dorid nudibranchs using accumulated data. The study provides insight into the most recent common ancestor of the nudibranch group, a complicated subject because of the lack of fossil records. Therefore, the manuscript is potentially considered for publication in the journal. However, there are some points in the manuscript that the authors should check and improve before the final decision of the editor.

- The first thing the authors should notice is the novelty of the study. Actually, the findings of the manuscript could be found elsewhere in the previous study. Thefore, the authors should add more information to highlight their novel points in the study.

- The findings of the current study mainly relied on the phylogeny of dorid nudiranch. However, it should be noted that the investigation of dorid nudibranch phylogeny is a complicated subject as tree topology is varied if different markers or approaches are used. Therefore, in addition to a few genes, larger data such as the mitogenome and transcriptome have been used for nudibranchs and may provide better resolutions. Some studies have been published, and the authors should refer to the manuscript. Also, in tree construction, different approaches are used, such as maximum likelihood and Bayesian inference. Therefore, the authors could consider adding a tree built by Bayesian inference to see similarities and differences between the two methods.

- As the author stated in the manuscript, “much of the trait data employed here lacks species-level resolution and might instead reflect broader patterns.” One of the key traits of the dorid nudibranch is the structure of the gill and gill cavity. For these characteristics, Korshunova et al. (2020) have already discussed them in detail. Even though in the present study, the authors did not include the gill and gill cavity for analyses, discussion about these in relation to the phylogeny of the dorid nudibranch is necessary.

- Some citations are missing from the reference, such as “Hallas et al. 2017." The authors should carefully check and include them.

Reviewer #2: This manuscript describes the authors’ use of publicly available data to construct a phylogeny and trace the evolution of feeding, color pattern and chemical defense (whether sequestered, secondarily modified or synthesized de novo) within dorid nudibranchs. Overall, it is nice to see publicly available data being used to tackle interesting questions about the evolution of color patterns in nudibranchs, which is an area very much in its infancy. However, there are certainly limitations to using these data within dorids since to date, no previously published phylogenetic analysis of Sanger-generated markers has successfully resolved dorid phylogeny (and this problem has yet to be resolved—if possible—with high throughput DNA sequencing data). The lack of phylogenetic resolution seems to be a huge hurdle to tracing evolution of traits within this group. Based on their analyses, the authors claim that the most recent common ancestor to dorids likely fed on sponges, possessed complex color patterns, and sequestered defensive metabolites from their sponge prey. In it’s present form, this manuscript is not ready for publication, but I encourage the authors to revise and resubmit. Generally, there are some definitions that need to be better defined with respect to the traits explored in the paper, and more details need to be included for the methods and results. There are also parts of the paper that are factually inaccurate and need correction.

I have included some general suggestions for the authors on ways to improve the manuscript, as well as more specific suggestions according to specific line in the manuscript.

Some thoughts on chemical defense: Chemical defense within sea slugs is not my primary of expertise, but it is unclear to me whether there is a distinction to be made between the use of secondary metabolites vs acid secretion within the context of heterobranch “chemical defense”. I have interpreted this distinction from existing literature. Are both of these considered chemical defense in the context of this study? I think the former are far more common within nudibranchs, and the latter far more common in the sister taxon, Pleurobranchomorpha (see Wägele et al., 2017), but acid secretion is used for defense in at least some nudibranchs. However, perhaps this is an outdated or oversimplified artificial distinction (?). Are the two phenomena related in some way, e.g. developmentally? If there is a distinction to be made, then I think it would make the paper more robust to make/address that distinction in the introduction or discussion. In other words, how chemical defense is defined will have an impact on our understanding of how the mechanisms have evolved within sea slugs. Similarly, does secondary modification imply that species that do this are also sequestering from their prey? If so, then shouldn’t the taxa that are considered to be secondary modifiers also sequesters? If a species synthesizes de novo, does that mean that by definition they are incapable of secondary modification? or can they be considered to “secondarily modify” if they modify a molecule that they synthesized themselves? It would better serve this work to clearly define what each of these mean.

Lines 36-7 contain what appears to be an error, stating that Doridoidei “is the most diverse heterobranch group.” This depends on what the author means by “diverse”. The most speciose heterobranch group is most definitely the terrestrial Stylommatophora, but perhaps the author is referring to diversity of morphology? or perhaps meant to say the most diverse/ or speciose marine heterobranch group. There are many ways to resolve this.

Line 44: I “species poor” might be a better alternative to “less-diverse” sister group

Lines 46-47: There are so many species of dorids that do not feed on sponges. Is there a citation to support that most are thought to feed exclusively on sponges? or some basic numbers you could include here? As written this feels like an oversimplification. I can think of many dorids that feed on bryozoans, barnacles, tunicates, spirorbid worms, entoprocts, even ophiuroids. Perhaps a better distinction to be made here, which I think you started to do is generalist feeders vs specialists.

Line 48: I suggest to add “marine” in front of “heterobranchs” or find a citation that suggests this is applicable for terrestrial heterobranchs (if it is!). Terrestrial heterobranchs vastly outnumber marine ones, but these two citations apply to marine lineages.

Line 68: substitute “often” for “typically” and is there a reference you can cite here?

Line 75-77: These citations focus on chromodoridids, Knutson and Gosliner 2022 is another, more recently published example of distantly related species sharing similar color patterns, but from within a group of non sponge feeding, non-chromodoridid dorids

Line 84: Presumably this has been studied within marine fishes, which are speciose and also feature many diverse color patterns? If so, then this should be touched upon here, or if not, then this could be used as an extra point for how little is known in marine systems, otherwise, specify marine invertebrates

Lines 85-86: Perhaps using molecular methods, but authors have previously discussed evolutionary scenarios for evolution of prey and metabolite sequestration vs synthesis de novo, see for example Cimino and Ghiselin 1999 and references therein. It would be good/appropriate to acknowledge this historical context since this has been a question of interest for some time.

Methods: “Mining” seems vague here. “Data mining” often implies computer automation, but if you automated this process, then there are details that are missing from within the methods here. I suggest modifying the language with to language without misleading connotations

Lines 104-5: Did you encounter multiple representatives that may have had “sufficient genetic data to support phylo. reconstruction”? if so, how did you choose which taxa/sequences to use?

Line 125: brackets- in my copy they are parentheses, not brackets

Table 1: While a supplementary document is included that lists the references used to complete the trait data in this table, it is not clear in many cases which reference was used to specify trait data for a particular taxon. It is essential that the authors link the specific traits to the actual sources for the work to be fully transparent and useful to future researchers—perhaps this can be done with superscripts or subscripts, if allowed by the journal formatting.

Phylogenetic and ancestral state reconstruction.

The authors chose to only include one phylogenetic reconstruction method (maximum likelihood, ML with bootstraps) for their phylogeny, though it is generally accepted within systematic studies to additionally run Bayesian Inference and present the support values from each method. Occasionally these methods corroborate each other and other times they may show conflict. Given the lack of resolution of this phylogeny, it seems particularly appropriate to include results from Bayesian Inference. Authors should perform Bayesian Inference, ensuring convergence of chains, and present these results alongside the ML results, addressing any conflicts or consistencies between methods.

I have minimal experience with Ancestral State Reconstruction, so I cannot directly comment on the methodology employed by the authors for this purpose.

Lines 161/2: Again, I’m less not very familiar with the details of using BayesTraits, but certainly some details of the analyses have been left out of this section? MCMC should have parameters that are set, right? and you need to have reached convergence for the results to mean anything… I couldn’t find it mentioned in the paper that convergence was reached for this analysis.

Line 167: replace “resolve” with “reconstruct”

Line 176: Please cite here the studies that find Actinocyclidae as sister to all other Doridoidei. E.g. Kurshonova et al 2020 does not demonstrate that Actinocyclidae is the sister group to the rest of Doridoroidei. If you look closely at their tree, most nodes at the backbone of dorids have no support (they neither listed the actual support, nor collapsed the unsupported nodes).

The Hallas paper presented many differing topologies, in part to show how different alignment methods impacted the resulting trees. What is more interesting, is that this lack of phylogenetic resolution at the backbone of dorids likely reflects rapid diversification and ancient incomplete lineage sorting, etc that makes this phylogeny particularly difficult to resolve.

Lines 184/5: To be fair, some of these genera have been described or resurrected since these two older papers were published.

Line 235-238: It is not clear what this sentence or the reference is meant to add here to this section. please rework

Line 245: Technically, the species of Gymnodoris that you’ve included in your phylogeny (G. ceylonica) does not feed on nudibranchs, it is documented to feed on other lineages of sea slugs. Remove “(nudibranchs)”

Line 283: This sentence contradicts your dataset. Table 1 shows that Lecithophorus, Colga and Vayssierea (which, less face it…it’s a polycerid) are all uniform in color pattern, and I can think of other polycerids as well. “entirely absent” is not true in the case of polycerids.

References: The authors must double check the entire manuscript to ensure that all references cited in the text appear in this section. For example, I noticed that Hallas et al. 2017 was cited a few times, but it is not listed in this section.

Figures: The full tif file of images (Fig 1) looks great. However, the downsampled image that downloaded as part of the reviewer pdf is way too dark and compromises the messages that the authors are trying to make about color patterns. To the authors- make sure to work with the editors to make sure that the final published version is still visually acceptable

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PLoS One. 2025 Apr 2;20(4):e0317704. doi: 10.1371/journal.pone.0317704.r003

Author response to Decision Letter 1

13 Dec 2024

Response to Reviewers

We thank both reviewers for their helpful revisionary suggestions and comments that have significantly improved this manuscript. We provide a detailed response to each of the reviewer’s comments below and have made the corresponding changes throughout the manuscript.

Reviewer One

C1_1: The first thing the authors should notice is the novelty of the study. Actually, the findings of the manuscript could be found elsewhere in the previous study. Therefore, the authors should add more information to highlight their novel points in the study.

R1_1: We thank the reviewer for their concerns about novelty, and we certainly want to make sure that we’re communicating the novelty and importance of this work. We agree, in part, that a multi-gene phylogeny for dorids has been presented in earlier papers (both by Korshunova et al. 2020 and Hallas et al. 2017), but in fact, we do add new data (both taxa and loci) to this phylogeny. We would argue, however, that the most significant and novel elements of this work are 1) the compilation of one of the most extensive trait datasets to date for dorid nudibranchs and 2) a first exploration of the evolution of these traits over deep time with insights into evidence (or lack thereof) of correlated evolution amongst these traits. It is important for the reviewer to know that this work stems from an MSc project that took place during the pandemic where we were unable to generate new data. I hope the past few years have shown us (the scientific community) the power of compiling and analyzing existing data in new and exciting ways.

C1_2: The findings of the current study mainly relied on the phylogeny of dorid nudibranch. However, it should be noted that the investigation of dorid nudibranch phylogeny is a complicated subject as tree topology is varied if different markers or approaches are used. Therefore, in addition to a few genes, larger data such as the mitogenome and transcriptome have been used for nudibranchs and may provide better resolutions. Some studies have been published, and the authors should refer to the manuscript. Also, in tree construction, different approaches are used, such as maximum likelihood and Bayesian inference. Therefore, the authors could consider adding a tree built by Bayesian inference to see similarities and differences between the two methods.

R1_2: We agree with the reviewer that genome-wide data will be absolutely critical for resolving nudibranch phylogeny (if at all). We do discuss recent efforts to sequence exons, ultraconserved elements (UCEs) and whole transcriptomes on lines 215-224 of the combined Results and Discussion. We did however overlook the inclusion of recent mitogenome work and one transcriptome paper, which we now include in this section. We also now include the Bayesian phylogeny as this was also suggested by Reviewer 2, please see Figure S1 and lines 156-158 and 200-207 in the main text.

C1_3: As the author stated in the manuscript, “much of the trait data employed here lacks species-level resolution and might instead reflect broader patterns.” One of the key traits of the dorid nudibranch is the structure of the gill and gill cavity. For these characteristics, Korshunova et al. (2020) have already discussed them in detail. Even though in the present study, the authors did not include the gill and gill cavity for analyses, discussion about these in relation to the phylogeny of the dorid nudibranch is necessary.

R1_3: We thank the authors for their comment, and we appreciate the wonderful body of morphological work that was presented in Korshunova et al. 2020. We have not discussed the gill and gill cavity work here since it doesn’t explicitly link to our aims/objectives in trying to understand the intimate link between prey preference, chemical defence and colour pattern variation. It seems logical to link these traits given that both metabolites (chemicals) and pigments (colour) can be sequestered from prey, but we don’t believe that gill morphology is a relevant trait to include here. Again, we absolutely agree that the paper by Korshunova et al. 2020 provides an incredibly comprehensive overview of gill and gill cavity morphology and evolution, and we commend these authors for their work, but we don’t believe this needs reiterating in our manuscript.

C1_4: Some citations are missing from the reference, such as “Hallas et al. 2017." The authors should carefully check and include them.

R1_4: Thank you for noticing this. We have since carefully revised the references.

Reviewer Two

C2_1: Some thoughts on chemical defense: Chemical defense within sea slugs is not my primary of expertise, but it is unclear to me whether there is a distinction to be made between the use of secondary metabolites vs acid secretion within the context of heterobranch “chemical defense”. I have interpreted this distinction from existing literature. Are both of these considered chemical defense in the context of this study? I think the former are far more common within nudibranchs, and the latter far more common in the sister taxon, Pleurobranchomorpha (see Wägele et al., 2017), but acid secretion is used for defense in at least some nudibranchs. However, perhaps this is an outdated or oversimplified artificial distinction (?). Are the two phenomena related in some way, e.g. developmentally? If there is a distinction to be made, then I think it would make the paper more robust to make/address that distinction in the introduction or discussion. In other words, how chemical defense is defined will have an impact on our understanding of how the mechanisms have evolved within sea slugs. Similarly, does secondary modification imply that species that do this are also sequestering from their prey? If so, then shouldn’t the taxa that are considered to be secondary modifiers also sequesters? If a species synthesizes de novo, does that mean that by definition they are incapable of secondary modification? or can they be considered to “secondarily modify” if they modify a molecule that they synthesized themselves? It would better serve this work to clearly define what each of these mean.

R2_1: We thank the reviewer for providing this important insight about nudibranch chemical defence. In light of this comment, we have simplified the trait data to include only ‘Sequester’ or ‘Synthesize’. Our motivation for making this change is, in part, because after having re-reviewed the literature and our data we simply don’t have enough information to confidently assign ‘Secondary Modification’ to many groups. We do have this information for some (the 6 taxa which were marked as such in the first version of the manuscript) but that information is lacking for others, and as the reviewer points out, it’s possible that even those taxa that de novo synthesize still secondarily modify their molecules. We have updated the trait data and corresponding figures (Figure 3) accordingly (and see lines 117-119). We appreciate the comment about acid secretion, although we don’t feel there’s enough information available to make this distinction here. The prevailing mechanism is the use of secondary metabolites.

C2_2: Lines 36-7 contain what appears to be an error, stating that Doridoidei “is the most diverse heterobranch group.” This depends on what the author means by “diverse”. The most speciose heterobranch group is most definitely the terrestrial Stylommatophora, but perhaps the author is referring to diversity of morphology? or perhaps meant to say the most diverse/ or speciose marine heterobranch group. There are many ways to resolve this.

R2_2: We thank the reviewer for noticing this oversight and we have revised the text on lines 36-38 to read:

“The Doridoidei infraorder (herein referred to as dorids) is one of the most speciose nudibranch groups, comprising over 2,000 species”

C2_3: Line 44: “species poor” might be a better alternative to “less-diverse” sister group

R2_3: We have revised the text on lines 44-45 to read:

“For instance, Bathydoridoidei, the comparably species poor sister group to Doridoidei“

C2_4: Lines 46-47: There are so many species of dorids that do not feed on sponges. Is there a citation to support that most are thought to feed exclusively on sponges? or some basic numbers you could include here? As written this feels like an oversimplification. I can think of many dorids that feed on bryozoans, barnacles, tunicates, spirorbid worms, entoprocts, even ophiuroids. Perhaps a better distinction to be made here, which I think you started to do is generalist feeders vs specialists.

R2_4: We thank the reviewer for this suggestion and we agree that our original statement wasn’t accurate. We have since revised the text below on lines 46-47 and include a reference to the original paper that proposed this specialization:

“...while most members of the Doridoidei are thought to have more specialized diets (Faulker & Ghiselin 1983)”

C2_5: Line 48: I suggest to add “marine” in front of “heterobranchs” or find a citation that suggests this is applicable for terrestrial heterobranchs (if it is!). Terrestrial heterobranchs vastly outnumber marine ones, but these two citations apply to marine lineages.

R2_5: We have the revised the text to specify “marine heterobranchs” as suggested.

C2_6: Line 68: substitute “often” for “typically” and is there a reference you can cite here?

R2_6: We have made this substitution and added a reference here.

C2_7: Line 75-77: These citations focus on chromodoridids, Knutson and Gosliner 2022 is another, more recently published example of distantly related species sharing similar color patterns, but from within a group of non sponge feeding, non-chromodoridid dorids

R2_7: We thank the reviewer for this helpful suggestion, we have now added Knutson and Gosliner 2022 on line 77.

C2_8: Line 84: Presumably this has been studied within marine fishes, which are speciose and also feature many diverse color patterns? If so, then this should be touched upon here, or if not, then this could be used as an extra point for how little is known in marine systems, otherwise, specify marine invertebrates

R2_8: We thank the reviewer for this helpful suggestion. There hasn’t been much work, if any, linking these traits in marine fishes either (like the polychromatic and venomous blennies, for example), but we have modified this sentence to satisfy the next comment from the reviewer. Please see below.

C2_9: Lines 85-86: Perhaps using molecular methods, but authors have previously discussed evolutionary scenarios for evolution of prey and metabolite sequestration vs synthesis de novo, see for example Cimino and Ghiselin 1999 and references therein. It would be good/appropriate to acknowledge this historical context since this has been a question of interest for some time.

R2_9: We appreciate this feedback and agree that this sentence needed revising. We have since revised as follows on lines 85-87:

“Prey preference, chemical acquisition and colour pattern have been described and studied across several nudibranch groups (see above), but no study has investigated the evolution of these traits in tandem, especially in a phylogenetic context.”

C2_10: Methods: “Mining” seems vague here. “Data mining” often implies computer automation, but if you automated this process, then there are details that are missing from within the methods here. I suggest modifying the language with to language without misleading connotations

R2_10: We generally disagree with this comment from the reviewer and have used, and seen others use, ‘mining’ in a manual context. That being said, it’s possible that others may misinterpret its usage as well and thus we substitute ‘compiling’ for ‘mining’ on line 102.

C2_11: Lines 104-5: Did you encounter multiple representatives that may have had “sufficient genetic data to support phylo. reconstruction”? if so, how did you choose which taxa/sequences to use?

R2_11: Yes there were multiple individuals per species and also multiple species per genus available on GenBank, however, we chose those that had the best genetic coverage. For example, if there were two species of Goniobranchus on GenBank but one of these species had only two loci available, then we prioritized the species that had three or more loci. We felt it was critically important to have as complete a genetic matrix as possible for this Doridina phylogeny.

C2_12: Line 125: brackets- in my copy they are parentheses, not brackets

R2_12: This has been changed to parentheses on line 148.

C2_13: Table 1: While a supplementary document is included that lists the references used to complete the trait data in this table, it is not clear in many cases which reference was used to specify trait data for a particular taxon. It is essential that the authors link the specific traits to the actual sources for the work to be fully transparent and useful to future researchers—perhaps this can be done with superscripts or subscripts, if allowed by the journal formatting.

R2_13: We thank the reviewer for this helpful suggestion and we have implemented these changes by linking specific trait data with corresponding references with superscripts in the table. Please see revised Table 1 and Supplementary File 1. Note, we also refined Table 1 to more explicitly highlight where trait data derives from family-level patterns, when we felt confident to do so (i.e. when there was no variation in traits across the rest of the confamilials and/or with accompanying morphological evidence).

C2_14: Phylogenetic and ancestral state reconstruction.

R2_14: We thank the reviewer for this suggestion. We have since implemented a Bayesian analysis in MrBayes and we include a figure in the supplementary materials (see Fig S1). The Bayesian tree has poor support at interior nodes and thus very few relationships amongst families are resolved. However, the same familial relationships are recovered in the Bayesian analysis as in the ML analysis reported in the main text. We include the relevant methods and results for this analysis in the manuscript, on lines 156-158 and 200-207.

C2_15: I have minimal experience with Ancestral State Reconstruction, so I cannot directly comment on the methodology employed by the authors for this purpose.

R2_15: The method employed here, using the corHMM package in R, was chosen because it allows you to test both equal rates and all-rates-different models, and it allows you to incorporate both missing data and multistate traits.

C2_16: Lines 161/2: Again, I’m not very familiar with the details of using BayesTraits, but certainly some details of the analyses have been left out of this section? MCMC should have parameters that are set, right? and you need to have reached convergence for the results to mean anything… I couldn’t find it mentioned in the paper that convergence was reached for this analysis.

R2_16: We thank the reviewer for noticing this as we did forget to specify the BayesTraits parameters, and the iterative process of this analysis, in the original version of the manuscript. We have now revised the Methods and Results accordingly to detail both parameters and significance. The revised text on lines 170-178 and 313-316 reads:

Methods:

“Lastly, we tested for correlated evolution among our three traits using an independent contrasts correlation model in an MCMC framework via a two-step process in BayesTraits v4.1.1 (Meade & Pagel 2024). First, we ran the ‘complex’ analysis, using a sample period of 1,000 with 1,010,000 iterati

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pone.0317704.s004.docx^{(25.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0317704.r004

Decision Letter 1

Satheesh Sathianeson

3 Jan 2025

Tracing the evolution of key traits in dorid nudibranchs

PONE-D-24-20562R1

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Reviewer #1: All comments have been addressed

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Reviewer #1: I appreciate the author's effort for the improvement of the manuscript. I have no further questions and recommend the manuscript for publication in PLOS One.

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PLoS One. doi: 10.1371/journal.pone.0317704.r005

Acceptance letter

Satheesh Sathianeson

PONE-D-24-20562R1

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

(TIF)

pone.0317704.s001.tif^{(663.9KB, tif)}

S1 File. Supplementary references (Table 1).

(DOCX)

pone.0317704.s002.docx^{(30.5KB, docx)}

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Data Availability Statement

Trait data is available within the manuscript and the sequence alignment employed for analysis is available on Dryad: 10.5061/dryad.8sf7m0d0m.

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PERMALINK

Tracing the evolution of key traits in dorid nudibranchs

Silvia Prieto-Baños

Kara K S Layton

Roles

Abstract

1. Introduction

2. Methods

Compiling genetic and trait data

Fig 1. Representatives of seventeen of the families included in this study, with image attributions in parentheses.

Phylogenetic and ancestral state reconstruction

3. Results and Discussion

Is the Doridoidei phylogeny resolved?

Fig 2. Multi-gene phylogeny (COI,16S,18S,28S,H3) for Doridoidei, constructed with IQ-TREE (1000 UF bootstraps, GTR + I + G) with families denoted by coloured boxes and bootstrap support values provided at each node.

How did chemical defence, prey preference and colour patterns evolve in Doridoidei?

Fig 3. Ancestral state reconstructions of (a) chemical acquisition, (b) prey preference and (c) colour patterns in Doridoidei, run with an equal rates (ER) model in corHMM in R.

4. Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Author response to Decision Letter 0

Decision Letter 0

Satheesh Sathianeson

Roles

Author response to Decision Letter 1

Decision Letter 1

Satheesh Sathianeson

Roles

Acceptance letter

Satheesh Sathianeson

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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