Morphological and molecular analyses of Anodontinae species (Bivalvia, Unionidae) of Lake Baikal and Transbaikalia

Abstract

The diversity and taxonomy of anodontine species in Lake Baikal and Transbaikalia region has been contentious since it is based on a typological species concept, the so called “Comparatory Method”. Using this method, six Comparatory anodontine species have been described for the study area as belonging to the genus Colletopterum. This genus was separated from Anodonta based on shell characteristics and further split into two subgenera, i.e. Colletopterum sensu stricto and Colletopterum (Piscinaliana). However, many authors do not recognize this separation maintaining all Colletopterum forms within Anodonta. The current study clarifies the taxonomy and systematics of Anodontinae in this region, using a combination of molecular, morphological and anatomical data. All previously recognized Comparatory forms are here recognized as a single species, i.e. Anodonta anatina.

Introduction

It is well established that the high intraspecific phenotypic shell variability of freshwater mussels (Bivalvia: Unionida) is intrinsically connected with environmental variables [1], [2], [3], [4], [5], [6]. Additionally, other factors, such as sex and ontogeny may also influence shell shape [7], [8], [9], [10]. Due to these reasons, the use of shell shape is limited to identify taxonomic units in freshwater mussels. In fact, this has been a persistent and contentious problem that resulted in exaggerating the extant number of freshwater mussel species [11], [12]. The Russian taxonomy for freshwater bivalves, the Typological Species Concept with the Comparatory Method’ (TCS-CM) [13], [14], uses mainly the arc of maximal convexity of the valve’s outline (AMCVO) and the index of convexity (ratio of shell width to shell height) for species delimitation [15], [16]. This system is known to inflate the number of species, especially those with high morphological plasticity of shells, which only reflect different ecophenotypes. In fact, recent studies using a multi-disciplinary approach, combining genetic analyses with morphological and anatomical studies, significantly reduced the number of TCS-CM in different freshwater mussel taxa. For example, Bolotov et al. [17] significantly reduced the number of recognized TCS-CM margaritiferid species, and the same occurred in the Unionidae family with several works by Klishko et al. [18], [19], [20], [21].

The most comprehensive current global classification of freshwater mussels only recognizes three species of the genus Anodonta for Russia, i.e. Anodonta anatina Linnaeus 1758, Anodonta cygnea Linnaeus 1758, and Anodonta beringiana Middendorff, 1851 [22]. However, a recent study placed A. beringiana in the genus Sinanodonta [23]. While A. anatina is widespread from Western Europe to Lake Baikal, inhabiting a wide variety of habitats, A. cygnea occurs in a more restricted range (from Western Europe east to the Ural Mountains) and only in lentic habitats [24]. Both species present great phenotypic plasticity having several hundreds of synonyms assigned for each in their global distribution, including in Russia [11], [12], [22]. Moreover, synonyms of each species are often mixed and misidentified [25]. Recognizing that most of these names correspond to ecophenotypes, Zhadin [5] listed seven Anodonta species and [11] three, including A. woodiana and A. beringiana that are now excluded from the Anodonta genus [23].

European-Siberian anodontine bivalves were placed in the genus Anodonta until the first half of the twentieth century [5], [26], [27], [28]. Later they were divided into two genera–Anodonta and Colletopterum [29], [30], [31], [32], based on the differences of umbo sculpture, shell surface and size of adult shells. The generic name Colletopterum was established by Bourguignat [29] for specimens collected in the River Danube (Bulgaria), named as C. praeclarum. Using the inflated TCS-CM, genus Colletopterum was subsequently recognized in the identification keys of freshwater mollusks [15], [16], [33], [34]. Nine species in the genus Colletopterum were recorded for Russia by Starobogatov et al. [15]. Later, Bogatov et al. [16] recognize 12 species for Russia, splitting them in two subgenera Colletopterum s. str. and Colletopterum (Piscinaliana) (Table 1; [16]). Recently, the species that were retained within Anodonta in these keys were synonymized with Anodonta cygnea and all species of Colletopterum (s.s.) and Colletopterum (P.) in the same keys were synonymized under Anodonta anatina [12], [22].

Table 1. Classification of the species corresponding to the TCS-CM genus Colletopterum Bourguignat, 1880.

Zhadin, 1938	Starobogatov et al., 2004	Bogatov et al., 2005	This Study
Genus Anodonta Lamark, 1799	Genus Colletopterum Bourguignat, 1880	Genus Colletopterum	Genus Anodonta
Subgenus Anodonta s. str.		Subgenus Colletopterum s. str.
Species	Species	Species	Species
A. anatina Linnaeus, 1758**	C. anatinum (Linnaeus, 1758)**	C. apollonicum (Bourguignat, 1880)*	A. anatina Linnaeus, 1758**
A. piscinalis Nilsson, 1822**	C. apollonicum (Bourguignat, 1880)*	C. baeri Bogatov, Starobogatov & Prozorova, 2005*
A. sedakovi Siemaschko, 1848******	C. depressum (Bourguignat, 1881)*	C. convexum (Drouët 1888)*
	C. nilssonii (Küster, 1842)**	C. milaschevichi Bogatov, Starobogatov & Prozorova, 2005*
	C. ponderosum (Pfeiffer, 1825)**	C. ostiarium (Drouët 1881)*
	C. piscinale (Nilsson, 1822)**	C. subcirculare (Clessin, 1873)*
	C. rostratum (Rossmaessler, 1836)**
	C. sorensianum (Dybowski, 1913)***	Subgenus Piscinaliana Bourguignat, 1880
	C. subcirculare (Clessin, 1873)*	Species
		C. anatinum (Linnaeus, 1758)**
		C. nilssonii (Küster, 1842)**
		C. piscinale (Nilsson, 1822)**
		C. ponderosum (Pfeiffer, 1825)**
		C. rostratum (Rossmaessler, 1836 **
		C. sorensianum (Dybowski, 1913)***

Distribution:

* Europe, European Russia

** European Russia, Siberia to Baikal Lake and Transbaikalia

*** Baikal Lake, Transbaikalia.

Because the Russian identification keys were based mainly in conchological and geographical factors, a combined methodology using genetic analyses with morphological and anatomical studies is needed to clarify the classification of Russian anodontine species assigned to Anodonta and Colletopterum genera.

Lake Baikal is the oldest and deepest lake in the world and is known by its high biodiversity and endemism [35]. However, the recorded diversity for freshwater mussels is extremely poor. In fact, up to the late 1990s, only a single anodontine species was recognized in Lake Baikal, i.e. Colletopterum (Ponderosiana) ponderosum sedakovi [32]. This number has been recently increased to five species [36] using the TCS-CM contentious methodology.

The aim of the present study was to revise the systematics and taxonomy of Anodontinae species from Lake Baikal and adjacent territory of Transbaikalia, using an integrative morphological, anatomical, and molecular approach. Furthermore, the study also aims to compare these findings with the classification used by TCS-CM, for anodontines from the study area and for those with a European distribution.

Materials and methods

Study area and sampling

A total of 139 anodontine specimens were collected from Lake Baikal, Lakes Ivan-Arachley, and River Lena basins, Russia, by scuba diving and seine in the coastal areas, during the summer of 2004 and autumn of 2016. (Fig 1). All specimens were measured to the nearest 0.1 mm including shell length (L), shell width (B), maximal shell height (H_m), and distance from umbo apex to the anterior shell margin (l). Additionally, the dimensions and shell characteristics of 29 specimens from European Russia were extracted from Zhadin [5] and Bogatov & Kijashko [33] for morphological and statistical analyses. Since freshwater mussels (Colletopterum sp.) are not rare, endangered or protected species, no permits are required for the collection of these mussels, in the study region. Additionally, field work did not involve any territory inside national parks or other protected areas. The map of the study area and collection site maps were built using QGIS 3.0 (Fig 1).

Fig 1. Map of the study area with anodontine specimens sampling sites (red circles).

A–location of Lake Baikal in Russia territory, B–Map of Lake Baikal and Transbaikalia region; Lake Baikal: 1 –Cherkalov Sor and 2 –Chyvyrkuy Bay; Baikal Basin (3 –River Selenga, 4 –Lake Torma, 5 –Lake Schuchje, 6 –Gusinoye, 7 –River Khilok); River Lena basin (8 –Lake Bol’shoe Eravnoe); Baikal-Lena basin (9 –Ivan-Arachley lake system including Lakes Shaksha, Arachley, Kergendu, Ivan and Tasey), closed lake-refuges (10 –Lake Kenon, 11 –Lake Arey).

Morphometry

Standard morphometric shell characters, i.e. B/H, H/L and l/L ratios were calculated according to Bogatov et al. [16] (Panels 1–3 in Fig 2). TCS-CM species identification was based on shell shape, B/H and l/L indexes, and standard curves of the Arc of Maximal Convexity of the Valve’s Outline (AMCVO) [16], [36] (Panels 4–6 in Fig 2). The age of each specimen was estimated by counting annual growth rings. Statistical analysis was performed using Microsoft Excel, 2010.

Fig 2. Shell measurements used for morphometry and species identification.

1 –lateral shell view showing: the distance from umbo apex to anterior shell margin (l) and shell maximal height (Hm) from umbo apex to ventral shell margin across maximal convex point of valves (mcp); 2 –dorsal shell view showing the mcp; 3 –anterior shell view and shell width (B) in the maximal convex point (mcp); 4 –arcs of maximal convexity of the valve outline of Baikalian species (Prozorova & Bogatov, 2006): A–C. ponderosum Pfeiffer 1825, B–C. anatinum Linnaeus 1758, C–C. piscinale Nilsson 1822, D–C. nilssonii Küester 1842, E–C. sorensianum Dybowsky, 1913; 5 –arcs of maximal convexity of the valve outline of Eurasian species: g– С. rostratum Rossmaessler 1836, h–C. ponderosum, i–C. anatinum, j– С. piscinale, k–C. nilssonii, l–C. sorensianum, and 6 –of European species: a–C. apollonicum (Bourguignat 1880), b–C. convexum (Drouet 1888), c–C. milaschevichi Bogatov, Starobogatov et Prozorova 2005, d–C. ostiarium (Drouet 1881), e–C. baeri Bogatov, Starobogatov et Prozorova 2005, f–C. subcirculare (Clessin 1873) according to Bogatov et al. (2005).

DNA extraction, PCR, sequencing and species identification

A total of 24 specimens collected from the study area, including at least 2 individuals per site and all previously identified TCS-CM species, were sampled for genetic analyses (Table 2). For comparison, six specimens from Ukraine were also included (Table 2). In detail, a small piece of foot tissue was clipped and stored individually in 96% ethanol. DNA extraction, PCR and sequencing conditions of F-type mtDNA cytochrome c oxidase subunit I (COI) followed Klishko et al. [20] (Annealing temperature 48-50ºC). Sequences were assembled using ChromasPro 1.7.4 (Technelysium, Tewantin, Australia). Alignment was performed in Bioedit 7.2.5 [37] including all newly sequenced individuals and all sequences from the most recent and comprehensive COI dataset for Anodonta anatina from Froufe et al. [25], with a total of 125 COI sequences. A haplotype network was constructed using TCS 1.21 [38] with a threshold of 95% and the output file was run through tcsBU [39]. Sequence divergences within the European haplogroup (uncorrected p-distance) were calculated using MEGA 7.0 [40]. Two distinct tools were used for this COI dataset to detect the number of Molecular Operational Taxonomic Units (MOTUs), and therefore the number of potential species. First, the Cluster Sequences tool implemented in BOLD 4 [41] was used and the generated BINs recognized as MOTUs. For the second, the 95% statistical parsimony connection limit using TCS 1.21 [38], was also applied.

Table 2. List of all individual specimens used for genetic analyses, collection sites and GenBank accession codes.

Species	Locality	Country	Code/GenBank	Comparatory species
Anodonta anatina	Lake Gusynoye	Russia	BIV3374/MH062766	Colletopterum anatinum
Anodonta anatina	Lake Gusynoye	Russia	BIV3375/MH062767	Colletopterum ponderosum
Anodonta anatina	Lake Schuchje	Russia	BIV3377/MH062768	Colletopterum nilssonii
Anodonta anatina	Lake Schuchje	Russia	BIV3378/MH062769	Colletopterum piscinale
Anodonta anatina	Lake Schuchje	Russia	BIV3379/MH062770	Colletopterum anatinum
Anodonta anatina	Lake Schuchje	Russia	BIV3381/MH062771	Colletopterum ponderosum
Anodonta anatina	Lake Torma	Russia	BIV3382/MH062772	Colletopterum anatinum
Anodonta anatina	Lake Torma	Russia	BIV3383/MH062773	Colletopterum nilssonii
Anodonta anatina	Lake Torma	Russia	BIV3384/MH062774	Colletopterum ponderosum
Anodonta anatina	Lake Torma	Russia	BIV3385/MH062775	Colletopterum piscinale
Anodonta anatina	River Selenga	Russia	BIV3387/MH062776	Colletopterum sorensianum
Anodonta anatina	River Selenga	Russia	BIV3388/MH062777	Colletopterum sorensianum
Anodonta anatina	Lake Big Eravna	Russia	BIV3392/MH062778	Colletopterum sorensianum
Anodonta anatina	Lake Big Eravna	Russia	BIV3393/MH062779	Colletopterum nilssonii
Anodonta anatina	Lake Big Eravna	Russia	BIV3394/MH062780	Colletopterum sorensianum
Anodonta anatina	Lake Big Eravna	Russia	BIV3395/MH062781	Colletopterum piscinale
Anodonta anatina	Lake Big Eravna	Russia	BIV3397/MH062782	Colletopterum anatinum
Anodonta anatina	Lake Big Eravna	Russia	BIV3399/MH062783	Colletopterum ponderosum
Anodonta anatina	Lake Baikal	Russia	BIV3401/MH062784	Colletopterum rostratum
Anodonta anatina	Lake Baikal	Russia	BIV3402/MH062785	Colletopterum ponderosum
Anodonta anatina	Lake Baikal	Russia	BIV3405/MH062786	Colletopterum anatinum
Anodonta anatina	Lake Baikal	Russia	BIV3406/MH062787	Colletopterum sorensianum
Anodonta anatina	Lake Baikal	Russia	BIV3408/MH062788	Colletopterum piscinale
Anodonta anatina	Lake Tasey	Russia	BIV0256/MH062765	Colletopterum ponderosum
Anodonta anatina	River Murafa	Ukraine	BIV0201/MH062760	Colletopterum ponderosum
Anodonta anatina	River Revna	Ukraine	BIV0202/MH062761	Colletopterum ponderosum
Anodonta anatina	River Revna	Ukraine	BIV0206/MH062764	Colletopterum subcirculare
Anodonta anatina	Pond in Dinotzky	Ukraine	BIV0204/MH062762	Colletopterum piscinale
Anodonta anatina	River Suchoy Tashlyk	Ukraine	BIV0205/MH062763	Colletopterum subcirculare
Anodonta anatina	River Vorsakla	Ukraine	BIV0199/MH062759	Colletopterum minimum

Results

General morphological features

Shell shape of collected specimens is highly variable, from ovate-rectangular and ovate-quadrangular to ovate-elongated with a curved or straight noticeable wing, on the dorsal margin (Table 3; S1–S6 Figs). Shell convexity varied from flattened to strongly convex. Periostracum is usually yellow-brown or green-yellow-brown; the nacre is white, yellowish or light blue. The anterior muscle scar is well imprinted and the posterior adductor scar is weakly visible, pseudocardinal and lateral teeth are absent.

Table 3. Shell metrics and TCS-CM identification of collected specimens.

Identification	Shell length	Shell width	Shell shape	B/H	H/L	l/L	AMCVO	TCS-CM
geography + TCS-CM	(mm)	(mm)						identification
C. sorensianum	28–80	1.5–1.9	ovate-quadrangular	0.35–0.44	0.64–0.82	0.19–0.25	l	C. sorensianum
			with wing				e	C. baeri
							f	C. subcirculare
C. nilssonii	48–87	1.6–2.2	ovate-quadrangular	0.41–0.47	0.55–0.77	0.21–0.27	k	C. nilssonii
			wing is low				c	C. milaschevichi
							d	C. ostiarium
C. piscinale	61–96	1.7–2.5	ovate-quadrangular or elongated	0.52–0.57	0.54–0.67	0.21–0.33	j	C. piscinale
			wing is low				b	C. convexum
C. anatinum	61–112	2.2–2.7	ovate-rectangular or quadrangular	0.52–0.57	0.52–0.62	0.21–0.33	i	C. anatinum
			wing almost unnoticeable				a	C. apollonicum
							b	C. convexum
							c	C. milaschevichi
							h	C. ponderosum
C. ponderosum	68–112	2.5–2.8	ovate-rectangular or elongated	0.58–0.63	0.52–0.63	0.18–0.27	h	C. ponderosum
			without wing				a	C. apollonicum
							b	C. convexum
							i	C. anatinum
C. rostratum	79–125	2.8–3.1	elongated oval, almost cylindrical	0.66–0.75	0.45–0.58	0.17–0.23	g	C. rostratum
			without wing				a	C. apollonicum

The umbo is wide and often eroded and does not project above the dorsal shell margin, its position from the anterior shell margin varies from 0.18 to 0.36% of the total shell length. Umbo sculpture of all examined specimens is double-looped [42] (Panels 1–4 in Fig 3) or with almost straight bars (Panels 5 and 6 in Fig 3), sometimes with discrete lines (Panels 7 and 8 in Fig 3). Umbo sculpture is strongly pronounced in some animals but weakly marked in others, regardless of age.

Fig 3. Umbo sculpture in Colletopterum species from studied region of Lake Baikal and Transbaikalia.

Soft body anatomy (or internal anatomy) is not dependent of shell shape and is similar in all specimens from the Lake Baikal, Baikal drainage and outside (Panels 1–3 in Fig 4). The mantle is an even cream color (Panels 1–3 in Fig 4: m). The inner and outer gills have the same shape and size, but the outer gills in gravid female have greater size than inner ones. Glochidia develop in the outer gills of female (Panels 2 and 3 in Fig 4.: ig, og). The foot is cream colored being darker or orange in the ventral part. The labial palps are well developed and tongue shaped (Panels 1–3 in Fig 4: f, lp). There are three mantle apertures: incurrent, excurrent and supra-anal (Panels 1 and 2 in Fig 4: ia, ea and sa). The papillae on the incurrent aperture are well developed, elongate-conical with swelling in base, with inner surface flat and convex on the outside. Sometimes papillae can be retracted due to preservation (Panels 4–9 in Fig 4: pia, pia1). The excurrent aperture lacks any papillae and has an unvarying pigmentation or pigmentation imitating papillae (Panels 6–8 in Fig 4: pg).

Fig 4. Inner anatomy features in Colletopterum specimens from Lake Baikal and Transbaikalia.

General view of soft body 4.1 –male, 4.2–4.3 –female with mature glochidia: m–mantle, ig–inner gills, og–outer gills, lp–labial palps, f–foot, ia–inner aperture, ea–excurrent aperture, sa–supra-anal aperture, aam–anterior adductor muscle, pam–posterior adductor muscle; 4.4: pia1 –retracted position and pia–normal position of papillae in the inner aperture, 4.5–4.8: pia–papillae shape of the inner aperture and pg–different pigmentation of excurrent aperture, 4.9 –papillae scaled-up.

Morphometry and species identification

Based on the TCS-CM and geographic distribution, six Comparatory species of Colletopterum (Piscinaliana) were identified in the study region: C. sorensianum, C. nilssonii, C. piscinale, C. anatinum, C. ponderosum and C. rostratum (Table 4, S1–S6 Figs). However, based only on the TCS-CM identification, i.e. the standard curves of the arc of maximal convexity of the valve’s outline (AMCVO) and morphometric indices, each separate TCS-CM species of Colletopterum (P.) from the studied region may also be identified as other TCS-CM species of Colletopterum s. str. from the European distribution (Table 3).

Table 4. TCS-CM species distribution of collected specimens.

Presence +, Absence -.

TCS-CM species	Sampling sites
	Baikal Lake		Baikal Lake Basin					Lena River Basin	Baikal Lake-Lena River Basin				Closed lakes
	1	2	3	4	5	6	7	8	9	10	11	12	13	14
C. sorensianum	+	+	+	-	-	-	-	+	-	+	-	-	-	-
C. nilssonii	+	+	-	+	+	+	-	+	+	+	+	-	-	-
C. piscinale	+	+	-	+	+	-	-	+	+	+	-	+	-	-
C. anatinum	+	+	+	+	+	+	+	+	+	+	-	+	-	+
C. ponderosum	+	+	-	+	+	+	-	+	+	+	+	-	-	-
C. rostratum	+	-	-	-	-	+	-	-	-	-	-	-	+	-

1 –Cherkalov Sor, 2 –Chyvyrkuy Bay, 3 –Selenga River, 4—Torma Lake, 5 –Shuchyje Lake, 6 –Gusinoe Lake, 7 –Khylok River, 8 –Bol’shoe Eravnoe Lake, 9–12 –Ivan-Arachley lakes system (including: 9 –Arachley Lake, 10 –Kergendu Lake, 11 –Tasey Lake, 12 –Ivan Lake), 13 –Kenon Lake and 14 –Arey Lake.

Morphometric analyses

Analyses of morphometric shell indexes B/H, H/L and l/L have shown significant variability and overlap in Colletopterum species from different localities of Lake Baikal, Transbaikalia and European Russia. This overlap can be substantial, even almost complete, in the TCS-CM species C. anatinum and C. piscinale (Table 3). The lower values of B/H were characteristic for all young individuals from all species and for C. sorensianum (including C. subcirculare and C. baeri). These values increased in C. nilssonii (including C. milaschewichi and C. ostiarium) and C. piscinale (including C. covexum) (Table 3; Panel 1 in Fig 5), with the higher values being obtained in C. ponderosum and C. rostratum (Table 3; Panel 1 in Fig 5). The values of H/L decrease in the reverse order (Panel 2 in Fig 5). Following the same order from C. sorensianum to C. rostratum, the position of the umbo (l/H) in the same species increases from C. sorensianum until C. anatinum and then decreased to C. rostratum (Panel 3 in Fig 5). The age of all examined specimens varied from < 1 year to 12 years, with shell length from 18 to 125 mm. The B/H showed a low variability range with an increasing trend with age (R² = 0.996) (Panel 4 in Fig 5). Individuals from distinct locations showed different growth rates, with the mean growth of individuals from all populations being shown in Panel 5 in Fig 5. Age is more closely connected with the B/H index values (R² = 0.996) than with shell length (R² = 0.928) with its higher accuracy reflecting its logarithmic dependence (R² = 0.964). In addition, B/H values increase with shell length in groups that have been identified as separate species (Fig 6). Shell shape, wing development and shell convexity change with shell length, corresponding to six different size-age group, in the analyzed TCS-CM species (Table 3).

Fig 5. Intraspecific variability and dynamic of mean values of morphometric shell indexes.

B/H– 5.1, H/L– 5.2, and l/L– 5.3 in Colletopterum species, range and change of mean values of B/H– 5.4 and shell length– 5.5 with increase of animal age from different localities in Lake Baikal, Transbaikalia and European Russia.

Fig 6. Change of shell shape and shell convexity with growth of shell length and index B/H in different age groups of TCS-CM Colletopterum species from different localities.

B–Lake Baikal, T–Transbaikalia, ER–European Russia: 1 –young and group of C. sorensianum including C. subcirculare and C. baeri, 2 –group of C. nilssonii including C. ostiarium and C. milaschevichi, 3 –group of C. piscinale including C. convexum, 4 –group of C. anatinum with transitional form C. apollonicum, 5 –group of C. ponderosum and 6 –group of С. rostratum.

All of the Comparatory species described from the study area correspond to at least another distinct TCS-CM species with a disjunct range (Table 3; S1–S6 Figs). While for C. piscinale and C. rostratum only a single additional species can be recovered (Table 3; S3 and S6 Figs), two species for C. sorensianum and C. nilssonii (Table 3; S1 and S2 Figs), three for C. ponderosum and even four additional species can be recovered in the case of C. anatinum (Table 3; S4 and S5 Figs). Additionally, specimens identified as C. anatinum, C. piscinale, and C. ponderosum can all be recognized as C. convexum with a total overlap of the curvatures of the AMCVO (Table 3; S3–S5 Figs). Furthermore, specimens described as C. anatinum can be assigned as C. ponderosum and vice-versa (Table 3; S4 and S5 Figs).

Molecular analyses

The aligned COI dataset presented a mean length of 578 bp with 21 polymorphic and 14 parsimony informative sets. No indels and no unexpected codons were observed in the corresponding amino-acid translation. TCS produced a single network shown in Fig 7. The 25 newly sequenced individuals from the study area, previously identified as six distinct TCS-CM species, resulted in five haplotypes (Dark Purple: Fig 7) all clustering within the previously recognized European A. anatina mitochondrial haplogroup (Purple: Fig 7) presenting a low intraspecific genetic diversity (p-distance = 0.6%). Eighty per cent of the individuals share a common haplotype with the remaining 20% representing 5 single private haplotypes. Moreover, the common haplotype is also shared with individuals from Poland and Sweden. From the six newly sequenced individuals from Ukraine, we retrieved four haplotypes also clustering within the European haplogroup (Fig 7). Both molecular species delineation methods applied in the COI dataset (Table 2) resulted in the identification of a single MOTU and therefore of a single species.

Fig 7. COI haplotype (TCS) network showing the relationships A. anatina haplotypes.

Colour codes correspond to the 5 haplogroups: Blue—Northwest Iberia; Green—Southcentral Iberia; Red Southwest Iberia; Purple—Europe; Yellow—Western Mediterranean. Within the European haplogroup, Russian haplotypes are represented in dark purple and Ukrainian in light purple. Circle size is proportional to the observed haplotype frequencies, white dots represent unobserved haplotypes.

Discussion

The present study represents the first comprehensive investigation of the TCS-CM Colletopterum species (Anodontinae) from Lake Baikal and adjacent territory of Transbaikalia. The integrative results here presented clearly show that all collected specimens belong to the species Anodonta anatina.

Molecular results additionally show that all analyzed specimens cluster within the European Anodonta anatina previously described mtDNA clade [25], with most individuals sharing haplotypes with specimens from Europe (Fig 7). Furthermore, no genetic subdivision was detected within the specimens from the studied region, discarding further division in subgenera and species. Both molecular species delimitation methods indicate a single MOTU, corroborating the existence of a single species, i.e. A. anatina. These results are further confirmed by the morphological and morphometric analyses.

According to Bogatov et al. [16], Colletopterum was separated from Anodonta by changes in shell size, umbo sculpture and shell surface. In the present study, all specimens were undoubtedly identified as a single species. The shell differences that A. anatina presents compared to other anodontine species within Anodonta are very weak. These slight differences do not support the separation of A. anatina and other Anodonta species (i.e., Anodonta cygnea and Western North American Anodonta) into distinct genera and should correspond to infrageneric differences. In fact, since anodontine species are known to present a high shell plasticity, these differences were even overlooked by the most comprehensive classification system carried by Haas [11], combining A. anatina with A. cygnea forms under a single species. Additionally, A. anatina clusters with A. cygnea and Anodonta nuttalliana in a monophyletic clade in the most recent molecular phylogeny [23].

According to Bogatov et al. [16], Colletopterum was separated into two subgenera based on shell characteristics, i.e presence/absence of a clear wing, but the same author noted that in mature or strongly convex specimens, this wing may be reduced. The present study revealed a considerable variability on the wing expression (S1–S6 Figs), without any correspondence to the two putative Colletopterum subgenera. Again, the data here presented discards the existence of a subdivision within the putative Colletopterum species, i.e within Anodonta anatina.

The main characteristics used to delimit each of the 12 TCS-CM Colletopterum species are the arc of maximal convexity of the valve’s outline (AMCVO) and the index of convexity (ratio of shell width to shell height) (Panels 2–4 in Fig 2) [16]. The present study shows that these features are not species specific. In fact, each TCS-CM species might be identified by the AMCVO as two-three, or even four different species (Panel 12—in S3 Fig) from both putative subgenera (S1–S6 Figs). No significant differences were found with regard to double-looped umbonal sculpture, soft part anatomy, and larval and adult shell morphology that supports the distinction of Colletopterum in two distinct subgenera (Panels 1–3 in Fig 4) [43].

It is well established that shell shape of unionoids is strongly influenced by both biotic and abiotic factors such as age, size, sex of the mussels, and temperature, hydrological, hydrochemical and trophic conditions [5], [6], [10]. Furthermore, many unionid and especially anodontine mussels, exhibit considerable intraspecific shell shape variation, which is often related with their occurrence in distinct habitats, shifts of metabolism at sexual maturity or even by changes in allometric growth and other physiological characteristics [10], [44], [45], [46], [47]. This influence in shell shape is also shown here for A. anatina, where B/H values increase with shell length. These values may be divided in six classes (Fig 6), each including 2–3 TCS/CM species that present an overlap of the B/H values. The same classes can also be recovered using the other two morphometric indexes H/L, and l/L values. However, these morphometric classes are composed of TCS-CM species from distant geographic ranges. Since no other independent morphological, anatomical or molecular character exhibit the same pattern, it clearly shows that these indexes are being influenced by environmental factors and should not be used to divide species. Furthermore, all collected young individuals were included in the C. sorensianum class group which indicates that age is clearly influencing not only the B/H but also the H/L, and l/L values (Figs 5 and 6). In fact, using all specimens across all TCS-CM species, the B/H index show very little variability with age, supporting the idea that shell shape is closely linked with growth (Fig 6). Furthermore, the sex of the animal might also influence shell shape in Anodonta [10], and consequently influencing the B/H, H/L, l/L morphometric indexes and AMCVO curves. According to Zieritz & Aldridge [10] A. anatina is dimorphic, with females being more inflated than males from the same size-age, due to the presence of swollen marsupia filled with glochidia during gravidity.

In summary, morphological, morphometric and molecular genetic data show that all TCS-CM species from both previously recognized subgenera of genus Colletopterum represent size-age class groups of one polymorphic species, i.e. Anodonta anatina, widespread in the Lake Baikal, Transbaikalia, Siberia and Europe.

Supporting information

S1 Fig. Shell shape and species identification according to TCS-CM of specimens identified as C. sorensianum.

S1.1 –holotype C. sorensianum (figure from Starobogatov et al., 2004: 127, Table 26, Fig 5–6), S1.2 –Lake Baikal, S1.3 –Lake Kergendu, S1.4- S1.6 –Lake Bol’shoe Eravnoe; S1.7 –C. subcirculare from River Don and S1.8 –C. baeri from River Volga (figure copy from Bogatov & Kijashko, 2016: Table III, figures 29–30).

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S2 Fig. Shell shape and species identification according to TCS-CM of specimens identified as C. nilssonii.

S2.1 –Cherkalov Sor and S2.2 –Chivyrkuy Bay of Lake Baikal, S2.3 –Lake Arachley, S2.4 –Lake Schuchje, S2.5 –Lake Bol’shoe Eravnoe, S2.6 –Lake Torma, S2.7 –Lake Gusinoye, S2.8 –Lake Kergendu, S2.11 –Moscow region (№ 1, ZISP), S2.9 –C. milaschevichi from River Volga (holotype № 1, ZISP) and S2.10 –C. ostiarium from River Dnieper (№ 7, ZISP) (figures 6.11, 6.9–6.10 reproduced from Bogatov & Kijashko, 2016: Table II-III, fig. 26, 31–32).

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S3 Fig. Shell shape and species identification according to TCS-CM of specimens identified as C. piscinale.

S3.1 –Lake Gusinoye, S3.2 –Lake Torma, S3.3- S3.5 –Lake Bol’shoe Eravnoe, S3.6 –Lake Kergendu, S3.7 –Lake Arachley, S3.8 –Lake Ivan, S3.9 –Cherkalov Sor of Lake Baikal, S3.10 –River Volga (ZISP, figures reproduced from Bogatov & Kijashko, 2016: Table III, fig. 35: j).

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S4 Fig. Shell shape and species identification according to TCS-CM of specimens identified as C. anatinum from different localities.

C. anatinum: S4.1- S4.2 –Lake Gusynoe, S4.3 –Lake Arey, S4.4 –Lake Schuchje, S4.5 –Lake Torma, S4.6 –Lake Kergendu, S4.7 –Lake Bol’shoe Eravnoe, S4.8 –Lake Ivan, S4.9 –Lake Arachley, S4.10 –Cherkalov Sor, Lake Baikal and S4.12 –River Ivitza, Tverskaya region (collection of the Institute of Biology and Soil Science Far East Russian Academy of Sciences, Vladivostok); S4.11 – С. convexum from River Don (collection of the Zoological Institute Russian Academy of Sciences, Saint-Petersburg). Figures 8.11–8.12 reproduced from Bogatov & Kijashko, 2016: Table II-III, fig. 27 and 34).

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S5 Fig. Shell shape and species identification according to TCS-CM of specimens identified as C. ponderosum.

S5.1- S5.2 –Cherkalov Sor, Lake Baikal, S5.3 –Lake Schuchje, S5.4 –Lake Torma, S5.5 –Lake Gusinoye, S5.6, S5.12 –Lake Bol’shoe Eravnoe, S5.7, S5.9, S5.11 –Chyvyrkuy Bay of Lake Baikal, S5.8, S5.10 –Lake Arachley, S5.13 –Lake Kergendu, S5.14 –Lake Tasey, S5.15 –Lake Uzminskoye Lake, European Russia (№ 36, ZISP); S5.16 –C. apollonicum from Lake Appolonya, Southern Europe (№ 1, ZISP). Figures 9.15–9.16 reproduced from Bogatov & Kijashko, 2016: Table II-III, fig. 33 and 28.

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S6 Fig. Shell shape and species identification according to TCM-CS of specimens identified as C. rostratum.

S6.1 –Lake Baikal, S6.2 –Lake Gusinoye, S6.3 –Lake Kenon, S6.4 –Germany (№ 1, ZISP), S6.5 –River Ivitza, European Russia. Figures S6.4 and S6.5 reproduced from Bogatov & Kijashko, 2016: Table III, fig. 36).

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Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

Fundação para a Ciencia e tecnologia (FCT) funded the genetics work under project PTDC/AGRFOR/1627/2014. Research grants from the same institution were awarded to MLL (SFRH/BD/115728/2016) and EF (SFRH/BPD/108445/2015).