The mitochondrial genome of Bothriocyrtum californicum (O. Pickard-Cambridge, 1874) (Araneae, Halonoproctidae) from ultraconserved elements (UCEs)

Abstract

The mitochondrial genome of Bothriocyrtum californicum (O. Pickard-Cambridge, 1874) has been completely sequenced and annotated for the first time. This trap-door spider is found in California, United States. We used SPAdes to assemble contigs and the mitogenome was extracted from the resulting contigs. The mitogenome was annotated using MITOS2 and by comparing to other species in the order Araneae. The mitogenome is 14,081 base pairs, and includes 22 transfer RNA (tRNA) genes, 13 protein coding genes (PCG), 2 ribosomal RNA (rRNA) genes, and a control region. This is the first sequenced and annotated mitogenome for this species, genus, and the family Halonoproctidae.

Introduction

Spiders (Araneae) are important terrestrial predators whose unique characteristics, habitats, behaviors, and predatory nature allow them to fill essential ecological roles (Li et al. 2022). To date, there are 53,485 classified species within 4,479 genera, and 139 families of the order Araneae (World Spider Catalog 2025). The family Halonoproctidae consists of 6 genera and 145 species (World Spider Catalog 2025), including Bothriocyrtum californicum (O. Pickard-Cambridge, 1874) (Figure 1), one of three species in the genus Bothriocyrtum Simon, 1891. Bothriocyrtum californicum, also known as the California trap-door spider, is 21.5 mm total length including chelicerae (Gertsch and Wallace 1936), and is a spider native to shrublands and deserts of Southern California in the United States (Ramírez et al. 2013), as well as Arizona in the United States and northern Baja California Norte in Mexico (iNaturalist, https://www.inaturalist.org).

Figure 1.

Image of a Bothriocyrtum californicum (O. Pickard-Cambridge, 1874) specimen. Photo courtesy of M. Hedin.

Here, the mitochondrial genome of B. californicum was annotated from sequence contigs resulting from untraconserved element (UCE) capture. With UCE sequence data it is common to also sequence mitochondrial DNA, especially the barcode gene cytochrome oxidase subunit I (COX1; e.g. Branstetter et al. 2021) and other high copy loci, which can then be used in standard phylogenetic analyses (e.g. Derkarabetian et al. 2019). This is the first mitogenome of the family Halonoproctidae and the genus Bothriocyrtum to be fully sequenced and annotated. The mitogenome may be used in further analyses to better understand the evolutionary history of B. californicum.

Materials and methods

An immature B. californicum sample was collected on January 17, 2021, in Riverside County near Mission Creek Reserve in California, USA (33.9969° N, 116,60670° W), by M. Hedin, R. Monjaraz-Ruedas, and B. Gibson (voucher number SDSU_MY4897). Voucher specimens are deposited in the San Diego State University Terrestrial Arthropods Collection (SDSU_TAC), curated by Dr. Marshal Hedin (mhedin@sdsu.edu). Genomic DNA from the specimen was extracted from leg tissues using the DNeasy Kit (Qiagen GmbH, Hilden, Germany). The extraction was then quantified using a Qubit Flex Fluorometer (Thermo Fisher Scientific) and quality checked using agarose gels. Ultraconserved element (UCE) library preparation was performed at SDSU using previously standardized methods for arachnids (Starrett et al. 2017). Target enrichment was performed using the myBaits UCE Spider 2Kv1 kit (Arbor Biosciences; Kulkarni et al. 2020). Libraries were sequenced using 150 bp, paired-end sequencing on an Illumina HiSeq 4000 at UC Davis DNA Technologies Core.

For de novo assembly of a rough draft mitogenome, we used the SPAdes genome assembler v4.0.0 (Prjibelski et al. 2020), then “fished” the scaffolds file using the 16S gene of the spider Cheliceroides longipalpus (GenBank accession MH891570; Chen et al. 2019) to isolate and retrieve a contig as a rough draft mitogenome. To locate the COX1 gene, we annotated the rough draft mitogenome using MITOS2 (Donath et al. 2019) on the Galaxy server (The Galaxy Community, 2022). To get a final, circularized mitogenome, we then did de novo assembly with NOVOPlasty v4.3.5 (Dierckxsens et al. 2017) using the COX1 gene as the seed and the rough draft mitogenome as the reference genome. We used MITOS 2 (Donath et al. 2019) on the Galaxy server (The Galaxy Community, 2022) to annotate the circularized mitogenome, then we rotated the circularized mitogenome to move COX1 to the first position (Table 1). Any genes not found by MITOS2 were manually located by referencing the 16 species used for phylogenetic analysis (see Figure 2), and using Li et al. (2022) to define the tRNA sequences of the same 16 species of spiders. The Proksee website (Grant et al. 2023) was used to manually annotate and visualize the genome.

Table 1.

Organization of Bothriocyrtum californicum (O. Pickard-Cambridge, 1874) (Araneae, Halonoproctidae) mitochondrial genome (NCBI GenBank accession number PQ043198).

Gene	Begin	End	Strand	Length	Intergenic nucleotidesa	Start	Stop
COX1	1	1536	+	1536	1	TTA	TAG
COX2	1538	2206	+	669	6	TTG	TAA
trnK	2213	2275	+	63	−13
trnD	2263	2314	+	52	−7
ATP8	2308	2460	+	153	−4	ATT	TAA
ATP6	2457	3122	+	666	0	ATA	TAA
COX3	3123	3908	+	786	−2	TTG	TAA
trnG	3907	3959	+	53	−3
ND3	3957	4292	+	336	−6	ATA	TAG
trnL2	4287	4337	−	51	−2
trnN	4336	4395	+	60	−15
trnA	4381	4433	+	53	−6
trnS1	4428	4478	+	51	−2
trnR	4477	4538	+	62	−15
trnE	4524	4578	+	55	−13
trnF	4566	4620	−	55	1
ND5	4622	6262	−	1641	−3	ATT	TAA
trnH	6260	6311	−	52	−6
ND4	6306	7599	−	1294	0	TTG	Tb
ND4L	7600	7858	−	259	−6	ATC	Tb
trnP	7853	7906	−	54	0
ND6	7907	8333	+	427	−2	TTG	Tb
CytB	8332	9471	+	1140	−8	TTG	TAG
trnS2	9464	9516	+	53	1
trnT	9518	9577	+	60	−16
ND1	9562	10470	−	909	−8	ATT	TAA
trnL1	10463	10521	−	59	2
16S	10524	11587	−	1064	−3
trnV	11585	11643	−	59	5
12S	11649	12339	−	691	−7
trnI	12333	12391	−	59	18
trnQ	12410	12474	−	65	0
Control Region	12475	12964	±	490	0
trnM	12965	13024	+	60	−10
ND2	13015	13942	+	928	6	ATA	Tb
trnW	13949	14003	+	55	−21
trnY	13983	14041	−	59	−6
trnC	14036	13	−	59	−13

^aNumbers indicate the number of nucleotides to the start of the adjacent gene. Negative values correspond to overlapping nucleotides between adjacent genes. ^bTermination codon completed via polyadenylation.

Figure 2.

A. Maximum likelihood analysis of 17 spider species and an outgroup based on the combined 13 protein coding genes: Wadicosa fidelis (KP100666; Wang et al. 2016), Oxytate striatipes (KM507783; Kim et al. 2016), Oxyopes sertatus (KM272950; Pan et al. 2016a), Habronattus oregonensis (AY571145; Masta and Boore 2004), Selenops bursarius (KM114573; Pan et al. 2016b), Cheiracanthium triviale (MN334527; Tyagi et al. 2020), Araneus angulatus (KU365988; unpublished), Loxosceles similis (MK425700; Kalapothakis et al. 2019), Pholcus phalangioides (JQ407804; unpublished), Parachtes romandialae (MN052923; Pons et al. 2019), Hypochilus thorelli (EU523753; Masta and Boore 2008), Phyxioschema suthepium (JQ407802; unpublished), Bothriocyrtum californicum (PQ043198), Calisoga longitarsus (EU523754; Masta and Boore 2008), Ornithoctonus huwena (AY309259; Qiu et al. 2005), Heptathela hangzhouensis (AY309258; Qiu et al. 2005), Liphistius erawan (JQ407803; unpublished), and the outgroup scorpion Tityus serrulatus (AY309258; Martins et al. 2015). Numbers on branches represent bootstrap support at that node. The accession numbers of the mitochondrial genome sequences for each species used in this analysis are listed after each species.

To confirm the phylogenetic position of B. californicum, the 13 protein coding genes of this species and the other 16 species of spiders (order Araneae) in NCBI and spread across the spider phylogeny of Li et al. (2022), plus the 13 protein coding genes of the outgroup scorpion, Tityus serrulatus (GenBank accession KR024030; Martins et al. 2015), were used to create a maximum-likelihood (ML) tree using RAxML version 8.2.12 (Stamatakis 2014). Each protein coding gene from all the spider species and the scorpion outgroup was translated to its amino acid sequence, and then amino acid sequences were aligned for each protein coding gene in Geneious Prime version 2024.0.5 (https://www.geneious.com) using MAFFT version 7.490 (Katoh and Standley 2013).

Results

The complete mitochondrial genome of B. californicum has 14,081 base pairs and consists of 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes, and 21 of the 37 genes were detected on the positive strand, while the remaining 16 were detected on the negative strand (Figure 2; Table 1). Read coverage for the specimen is in Supplemental Figure 1. An AT-bias was observed within the mitochondrial genome, displaying an AT-content of 69.2% (A = 31.7%, T = 37.5%, G = 21.7%, C = 9.1%). The 22 tRNAs are found throughout the coding region (Figure 3). The tRNA genes range from 51 bp (trnL2 and trnS1) to 65 bp (trnQ).

Figure 3.

The mitochondrial genome of the spider Bothriocyrtum californicum (O. Pickard-Cambridge, 1874) (Araneae, Halonoproctidae; NCBI GenBank accession number PQ043198). The circle represents the genome sequence with the genes to the outside representing their positions on the positive (+) strand, while those to the inner side represent the genes on the negative (-) strand. The gene orientation is represented by the square ends (5’) and arrow point (3’). The control region is presented as occurring on both strands.

Of the 37 genes identified, 25 overlaps were detected, the largest of these being a 21 bp overlap between trnW and trnY (Table 1). Seven intergenic nucleotide gaps of 1 bp or more were detected, the largest being an 18 bp gap between trnI and trnQ (Table 1). Only three pairs of adjacent genes lacked overlap and intergenic nucleotides (Table 1).

Of the 13 PCGs, three have the ATA start codon (ATP6, ND2, and ND3), three have the ATT start codon (ATP8, ND1, and ND5), one has the ATC start codon (ND4L), five have the TTG start codon (COX2, COX3, COB, ND4, and ND6), and one has the TTA start codon (COX1) (Table 1). Six of the PCGs have the TAA stop codon (COX2, COX3, ATP6, ATP8, ND1, and ND5) and three have the TAG stop codon (COX1, COB, and ND3) (Table 1). Four of the PCGs have the stop codon completed through poly-adenylation (ND2, ND4, ND4L, and ND6 terminate with T) (Table 1).

The phylogenetic analysis placed B. californicum among other mygalomorph spiders, including Calisoga longitarsis (Simon, 19891) and Phyxioschema suthepium Raven and Schwendinger, 1989 (Figure 2).

Discussion and conclusions

Bothriocyrtum californicum is the first species of its genus and the family Halonoproctidae, whose mitochondrial genome has been sequenced and annotated. The mitochondrial genome is 14,081 base pairs in length, and consists of 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. When compared to the three other mygalomorph species included in our phylogeny (Figure 2), the AT-bias of B. californicum is 69.2%, which is similar to the 69.8% AT-bias of Ornithoctonus huwena (GenBank accession AY305259; Qiu et al. 2005), and marginally more than the 67.4% of Phyxioschema suthepium (GenBank accession JQ407802; unpublished). The final mygalomorph species in our phylogeny, Calisoga longitarsus (GenBank accession EU523754; Masta and Boore 2008), has a much lower AT-bias at 64.0% even though it is more closely related to B. californicum than the other two mygalomorph species (Figure 2). However, we note that we have data for only these four species of mygalomorphs spiders and that these differences may be within the ranges of variability for mygalomorph spiders when more species have completed mitogenomes for a more robust comparison.

Consistent with Godwin et al. (2018) and Opatova et al. (2020), the phylogenetic placement of B. californicum is among other mygalomorph spiders. The results of this study will further contribute to the growing number of sequenced spider mitochondrial genomes, and will help further studies of phylogenetics of spiders as a whole.

Funding Statement

We thank the SD BRIN grant for supporting this research. “Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.” Research supported by the National Institute of General Medical Sciences of the NIH through the Innovative Programs to Enhance Research Training IPERT Grant 1R25GM125674-01 and the New Hampshire-INBRE Institutional Development Award P20GM103506. The Galaxy server that was used for some calculations is in part funded by Collaborative Research Center 992 Medical Epigenetics (DFG grant SFB 992/1 2012) and German Federal Ministry of Education and Research (BMBF grants 031 A538A/A538C RBC, 031L0101B/031L0101C de.NBI-epi, 031L0106 de.STAIR (de.NBI)). National Science Foundation grant (DEB1937725) for the funds allowing M. Hedin, R. Monjaraz-Ruedas, and B. Gibson to collect the specimen used in this study.

Ethical approval

Bothriocyrtum californicum is not an endangered species and research on this species requires no specific permissions or licenses.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The genome sequence data that support the findings of this study are openly available in

GenBank of NCBI at BioProject PRJNA1157860, SRA study SRX26025206, BioSample SAMN43524786, Run SRR30602577, and the accession number for the mitogenome is PQ043198.