Research Article |
Corresponding author: Marshal Hedin ( mhedin@mail.sdsu.edu ) Academic editor: Gergin Blagoev
© 2018 Marshal Hedin, Shahan Derkarabetian, Jennifer Blair, Pierre Paquin.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hedin M, Derkarabetian S, Blair J, Paquin P (2018) Sequence capture phylogenomics of eyeless Cicurina spiders from Texas caves, with emphasis on US federally-endangered species from Bexar County (Araneae, Hahniidae). ZooKeys 769: 49-76. https://doi.org/10.3897/zookeys.769.25814
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Morphological, mitochondrial, and nuclear phylogenomic data were combined to address phylogenetic and species delimitation questions in cave-limited Cicurina spiders from central Texas. Special effort was focused on specimens and cave locations in the San Antonio region (Bexar County), home to four eyeless species listed as US Federally Endangered. Sequence capture experiments resulted in the recovery of ~200–400 homologous ultra-conserved element (UCE) nuclear loci across taxa, and nearly complete COI mitochondrial DNA sequences from the same set of individuals. Some of these nuclear and mitochondrial sequences were recovered from “standard” museum specimens without special preservation of DNA material, including museum specimens preserved in the 1990s. Multiple phylogenetic analyses of the UCE data agree in the recovery of two major lineages of eyeless Cicurina in Texas. These lineages also differ in mitochondrial clade membership, female genitalic morphology, degree of troglomorphy (as measured by relative leg length), and are mostly allopatric across much of Texas. Rare sympatry was confirmed in Bexar County, where members of the two major clades sometimes co-exist in the same karst feature. Both nuclear phylogenomic and mitochondrial data indicate the existence of undescribed species from the San Antonio region, although further sampling and collection of adult specimens is needed to explicitly test these hypotheses. Our data support the two following species synonymies (Cicurina venii Gertsch, 1992 = Cicurina madla Gertsch, 1992; Cicurina loftini Cokendolpher, 2004 = Cicurina vespera Gertsch, 1992), formally proposed here. Overall, our taxonomy-focused research has many important conservation implications, and again highlights the fundamental importance of robust taxonomy in conservation research.
cave evolution, conservation, karst, mitochondrial by-catch, taxonomy, ultra-conserved element
The limestone cave and karst habitats of Texas are home to hundreds of endemic cave-obligate animal species, including many eyeless spider species. The spider subgenus Cicurella (genus Cicurina) includes 60 described species, almost all endemic to Texas caves (
Many authors have discussed the challenges of phylogenetic and taxonomic research in Texas cave Cicurina (
The special challenge of species delimitation in Texas cave Cicurina is exemplified by Cicurina venii. This federally endangered species is known only from a single adult female from the type locality (Bracken Bat Cave), the entrance to which has been buried since about 1990 (
An additional appeal of UCE-based phylogenomics is that the method has demonstrated applicability with degraded DNA (e.g., on “standard” museum specimens, without special preservation of DNA material). For example, hundreds or thousands of UCE loci have been captured from old bird museum specimens (
Specimens and/or DNA extractions were made available from multiple institutions and persons, including the Texas Memorial Museum (TMM), Texas Tech University (TTU), ZARA Environmental, and the American Museum of Natural History (AMNH) (see Acknowledgements). In total, we attempted to gather UCE data for 83 eyeless Cicurina specimens, six of which were “standard” museum specimens (without special preservation of DNA material, Suppl. material
Many specimens used in this study were immatures, but could be tentatively identified to species in a post hoc manner based on phylogenetic placement into genetic clades including adult specimens, bolstered by locality data (i.e., caves from which adult Cicurina specimens have been collected in the past, including type locations). The assumption of no sympatry is fundamentally important here (i.e., one eyeless Cicurina species per cave). No sympatry is the rule for these spiders (
For specimens preserved for DNA studies (preserved in high percentage ethyl alcohol at -80 °C), genomic DNA was extracted from leg tissue using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). For museum samples preserved in 70–80% EtOH we used standard phenol/chloroform extractions with a 24-hour incubation. All extractions were quantified using a Qubit Fluorometer (Life Technologies, Inc.) and quality was assessed via gel electrophoresis on a 1% agarose gel.
UCE data were collected in multiple library preparation and sequencing experiments. Up to 500 ng of genomic DNA was sonicated using a Covaris M220 Focused-ultrasonicator with treatment time of 60–65 s, Peak Incident Power of 50, 10% Duty Factor, and 200 cycles per burst. All museum samples were sonicated for 30 seconds using the same settings. Samples were electrophoresed on agarose gels to verify sonication success.
Library preparation followed
Target enrichment was performed on pooled libraries using the MYbaits Arachnida 1.1K version 1 kit (Arbor Biosciences;
Raw demultiplexed reads were processed using the Phyluce pipeline (
Individual UCE loci were imported into Geneious 10.1 (Biomatters Ltd.) for manual inspection. In particular, alignments with low % identical sites (less than 40%) were flagged for inspection. If exclusion of a single divergent sequence increased this value to > 75%, the locus was retained. Subsequently, all loci were inspected - individual sequences with large gaps in the core UCE region were excluded, and obvious alignment errors in flanking regions were manually adjusted.
Concatenated data matrices with 50% and 70% occupancy (i.e., for any given locus, sequences for at least 50 or 70% of samples needed for locus inclusion in the final dataset) were assembled for phylogenomic analyses. Maximum likelihood analyses of both matrices were conducted using RAxML version 8.2 (
Using a sample of eyed and eyeless Cicurina spiders from Texas and other US states,
For adult specimens used in UCE experiments we imaged genitalia using a Visionary Digital BK plus system (http://www.visionarydigital.com). Individual images were merged into a composite image using Helicon Focus 6.2.2 software (http://www.heliconsoft.com/heliconfocus.html). Because we did not have access to all specimens borrowed from TTU (some loans were DNA only), we were not able to image all adult specimens used in this study.
Suppl. material
Using molecular clock or mid-point rooting, all nuclear phylogenomic analyses result in recovery of two primary Texas eyeless Cicurina clades (Figures
Phylogenetic tree from RAxML analysis of 399 UCE loci, 70% (399_70) occupancy matrix. Bootstrap values below 75 not shown. Geography and morphology highlighted. Abbreviations: ME (= mostly elongate) spermathecae clade, R (= rounded) spermathecae clade. Further details regarding specimen codes and locations found in Suppl. material
We recovered mitochondrial COI sequences from UCE assemblies for all but two specimens (TMM_9790, TK_190994), with an overall matrix completeness above 97%. All UCE-derived COI sequences were in frame and lacked stop codons or ambiguities, and when directly compared to previously published Sanger data (e.g., from same cave or sometimes same specimen), were found to be identical (Figure
Maximum likelihood trees, rooted with the eyed taxon C. pampa, include an R clade, but the poorly supported placement of two ME sequences renders this latter group paraphyletic (Figure
Phylogenetic tree from RAxML analysis of COI mitochondrial data. Previously published sequences with corresponding GenBank numbers (AY#), some with cave location codes as in
Within the ME clade, nuclear phylogenomic relationships are structured geographically, with western, northeastern, and Bexar county groupings (Figure
Figure
We gathered nuclear UCE data for three specimens from Government Canyon Bat Cave (GCBC, the stated type locality of C. vespera), all of which fall into the C. madlaUCE genetic clade. This nesting of GCBC specimens inside a C. madla clade also applies to the COI dataset, which also includes two additional Sanger GCBC specimens (Figure
Morphology of adult C. madla with corresponding COI RAxML phylogeny. Previously published sequences with corresponding GenBank numbers (AY#), some with cave location codes as in
The R clade includes six described species with very similar genitalic morphologies (Figure
Cicurina loftini is found only in the Culebra Anticline KFR (Figure
We measured TI values for 49 total specimens, some of which were included in UCE experiments (Suppl. material
For 25 specimens representing the ME clade, all but two specimens have TI values above 2.04 (Figure
The spider genus Cicurina includes over 130 described species known from multiple regions in the northern hemisphere (
The capture-based DNA sequencing strategy implemented here provides a foundation for ultimately collecting nuclear phylogenomic data for all described eyeless Cicurina species in Texas. Collection of such data would be extremely important for testing existing species hypotheses, many of which are based on limited material and represent fundamentally weak hypotheses (see
Examples of eyed- and eyeless Cicurina taxa from the same Texas cave are numerous (e.g.,
Our discovery of divergent ME and R genetic lineages, with corresponding differences in degree of troglomorphy (Figure
The current study was partially constrained in two ways – first, we often used immature specimens, again because about 90% of collected eyeless specimens are immature (
We have shown that immatures of ME and R clade species from Bexar County differ in TI index (Figure
Within the ME clade, immature spiders from Bob Clark Cave (Bandera Co.) and Fern Cave (Medina Co.) represent a potentially undescribed sister species of C. madla, herein called C. cf. madla (Figure
Within the R clade, immature spiders from the “Oblate Pit Sexton” location clearly represent a second known population for the federally endangered species C. baronia (Figs
Both C. vespera and C. venii are single-site endemic species listed as US federally endangered (Figure
Conversely, the female morphology and TI index of the C. venii holotype (type location stated as Bracken Bat Cave) is like C. madla (
A key to resolving the above anomalous morphological and distributional data would have been successful collection of nuclear UCE data (or by-catch COI data) from the type specimens. We were unable to secure such data from the old holotype specimens. Given this lack of direct evidence for species status of the anomalous holotypes, we propose two alternative scenarios: 1)
Cicurina madla Gertsch, 1992: 109, figs 91–92.
Cicurina madla Cokendolpher, 2004: 42, figs 7, 40–47.
Cicurina madla Paquin & Dupérré, 2009: 28, figs 50–51, 134–135.
Cicurina venii Gertsch, 1992: 111, figs 95–96; syn. n.
Cicurina venii Cokendolpher, 2004: 52, figs 63–64; syn. n.
Cicurina venii Paquin & Dupérré, 2009: 52, figs 116–117; syn. n.
The adult morphology of a potentially undescribed sister species (C. cf. madla) remains unknown. ME clade members from the neighboring Stone Oak KFR (C. platypus, C. puentecilla) have different spermathecal morphologies (large, even-sized, rounded;
Female spermathecal morphology as described in
ME clade member known from approximately 25 caves or karst features in the Government Canyon, Helotes, UTSA and northern Culebra Anticline KFRs (Figure
The high TI index, elongate spermathecae holotype C. venii type specimen is either 1) actually from GCBC, but was mislabeled or placed into an incorrect vial, or 2) is actually from Bracken Bat Cave, and represents a further southern (but currently unknown) extension of the C. madla Culebra Anticline subclade (Figure
Cicurina vespera Gertsch, 1992: 111, figs 93–94.
Cicurina vespera Cokendolpher, 2004: 53, figs 65–66.
Cicurina vespera Paquin & Dupérré, 2009: 53, figs 118–119, 134.
Cicurina loftini Cokendolpher, 2004: 41, figs 5, 10, 37–39; syn. n.
Cicurina loftini Paquin & Dupérré, 2009: 27, figs 46–47, 134; syn. n.
Based on well-supported nuclear phylogenomic analyses (Figs
Female spermathecal morphology as described in
R clade member known from 16 cave or karst features in the Culebra Anticline KFR (Figure
The low TI index, rounded spermathecae holotype C. vespera type specimen is either 1) actually from Bracken Bat Cave, but was mislabeled or placed into an incorrect vial, or 2) is actually from GCBC, and represents a northern disjunct from most Culebra Anticline C. vespera populations, although we note that direct evidence for such an extension does not exist in the Government Canyon KFR (Figure
Male palpal morphology of C. madla and C. vespera (left palp, ventral view). Specimens from Stevens Ranch Trash Hole Cave (CIC_1745) and Robbers Cave (TX_241) from PP personal collection. Image of specimen TK_188519 from Joel Ledford. Abbreviations: CB = cymbium, T = tegulum, E = embolus. Scale bar: 0.5 mm.
We would like to thank James Cokendolpher, Kathy MacDonald, Heath Garner, and Caleb Phillips from the Museum of Texas Tech University, James Reddell from the Texas Memorial Museum, Jean Krejca and Rachel Barlow from ZARA Environmental, and Louis Sorkin and Lorenzo Prendini from the American Museum of Natural History. We also thank the many other persons who have collected Cicurina specimens ultimately used in this research (Suppl. material
Figure S1. SVD Quartets, 50 & 70% matrices
Figure S2. ASTRAL, 50 & 70% matrices
Figure S3. RAxML, 50% matrix
Figure S4. Relationships within C. baronia + C. bullis_C. neovespera clade
Table S1. Voucher and genetic information
Table S2. Troglomorphy Index data