Farallon Camel Cricket, Farallonophilus Cavernicolus

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Farallon Camel Cricket, Farallonophilus cavernicolus (Rentz, 1972) (Orthoptera: Rhapidophoridae), Relationship within the Ceuthophilinae Authors: Michael Valainis , Tim Andriese, Russell Bradley, Jaime Jahncke, and Jeffrey Honda Source: The Pan-Pacific Entomologist, 95(1) : 13-20 Published By: Pacific Coast Entomological Society URL: https://doi.org/10.3956/2019-95.1.13

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Downloaded From: https://bioone.org/journals/The-Pan-Pacific-Entomologist on 10 May 2019 Terms of Use: https://bioone.org/terms-of-use Access provided by University of California Davis THE PAN-PACIFIC ENTOMOLOGIST 95(1):13–20, (2019)

Farallon camel cricket, Farallonophilus cavernicolus (Rentz, 1972) (Orthoptera: Rhapidophoridae), relationship within the Ceuthophilinae

1 1 2 2 MICHAEL VALAINIS *, TIM ANDRIESE , RUSSELL BRADLEY , JAIME JAHNCKE 1 AND JEFFREY HONDA 1Department of Biological Sciences, San Jose State University, San Jose, California, U.S.A. 2Point Blue Conservation Science, 3820 Cypress Drive #11, Petaluma, California, U.S.A. *Corresponding author. E-mail: [email protected]

Abstract. Farallonophilus cavernicolus (Rentz, 1972) is a member of the family Rhaphi- dophoridae (camel crickets) endemic to the Farallon Islands. Rentz (1972) hypothesized that F. cavernicolus had characteristics between those of the genera Pristoceuthophilus Rehn, 1903 and Ceuthophilus Scudder, 1862 but closest to the genus Pristoceuthophilus. Currently Pris- toceuthophilus is placed in the tribe Pristoceuthophilini and Ceuthophilus in the tribe Ceutho- philini. Farallonophilus cavernicolus is currently placed within the Pristoceuthophilini (Cigliano et al. 2018). We tested this hypothesis and classifi cation by performing mtDNA phylogenetic analysis using collected samples and GenBank sequences. Our results indicate general agree- ment with current subfamily Ceuthophilinae (which contains the tribes Pristoceuthophilini and Ceuthophilini) relationships except that F. cavernicolus appears to be outside both the Pris- toceuthophilini and the Ceuthophilini. Our results suggest that the F. cavernicolus lineage diverged from the Pristoceuthophilini and Ceuthophilini roughly 8.9MYA, roughly 1.6 million years before the Pristoceuthophilini and Ceuthophilini diverged from each other. Keywords. Farallon Islands, Insect, Taxonomy, Ceuthophilinae, Pristoceuthophilini, Ceuthophilini.

INTRODUCTION The Farallon camel cricket, Farallonophilus cavernicolus (Rentz, 1972) has so far been found only on Southeast Farallon Island, 48 km west of San Francisco and 32 km south of Point Reyes, despite collection efforts along the California coast. Using ten morphological characters originally specifi ed by Hubbell (1936), Rentz (1972) felt that Farallonophilus Rentz, 1972 fell somewhere between Ceuthophilus Scudder, 1862 and Pristoceuthophilus Rehn, 1862, However, because F. cavernicolus had more characteristics in common with Pristoceuthophilus, he surmised that Farallonophilus is more closely allied to Pristoceuthophilus. These genera are currently placed within the subfamily Ceuthophilinae in the tribe Pristoceuthophilini, while other genera commonly found along the Pacifi c Coast of the United States (Ceuthophilus (Scudder 1862), Hemiudeopsylla (Saussure & Pictet, 1897), and Rhachocnemis (Caudell, 1916) are placed within the tribe Ceuthophilini (Cigliano et al. 2018). To date, no comparative studies have examined the relationships among these taxa, and there is no well- resolved phylogeny of Pristoceuthophilus (Conroy & Gray 2015). As part of a continuing effort to gain a better understanding of F. cavernicolus, we examined the phylogenetic relationship of this species with closely related taxa in the subfamily Ceuthophilinae using mtDNA sequence analysis to validate the hypothesis that F. cavernicolus is most closely allied to Pristoceuthophilus.

MATERIALS AND METHODS We surveyed several habitats within Oregon and California in search of the Farallon Island cricket and related species. While other species representing Pristoceuthophilini and Ceuthophilini were collected, i.e., Pristoceuthophilus, Hemiudeopsylla californianus Scudder, 1862, and Rhachocnemis validus Scudder, 1894, we did not fi nd F. cavernicolus, and this species appears to be limited to the Farallon Islands. We obtained specimens of F. cavernicolus during our study of the ecology of this cricket on Southeast Farallon Island, where it dwells in former surge caves that have been raised by tectonic uplift. A specimen of Pristoceuthophilus marmoratus (Rehn, 1903) was obtained from Dr. David Gray. In addition, we obtained barcode sequences from BOLD (Barcode of Life Data System) (Ratnasingham 2007). Specimens used in the phylogenetic analysis are listed in Table 1. Specimens of Raphidophoridae and Gryllidae were collected using rolled oats and dog food along baited trails by examining stations from dusk to approximately 24:00 hr. Individuals were identifi ed to at least to genus using morphological keys. Pristoceuthophilus samples were not identifi ed to species but were given an identifi cation code and used as a clade to compare against F. cavernicolus and other taxa. Voucher specimens are maintained at the J. Gordon Edwards Entomology Museum at San Jose State University. DNA extraction was done from a single leg using a DNeasy extraction kit following the kit protocol for animal tissues (Qiagen, Valencia, California). We amplifi ed and sequenced a total of 1273 bp using parts of two mtDNA genes: cytochrome c oxidase gene (COI) and nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5). These sequence regions were specifi cally used as sequence data, as closely related taxa sequences for these regions are found online and can be used for a more holistic comparison. Published primer sequences for each gene region amplifi ed were used. For COI, two regions were amplifi ed. The fi rst region (bar-code region) used primers as described from Folmer et al. (1994): 5’-GGTCAACAAATCATAAAGATATTGG-3’ (LCO1490 forward primer) 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’ (HC02198 reverse primer) The second set of COI primers used were from Simon et al. (1994) and Hafner et al. (1994), respectively: 5’-GGAGGATTTGGAAATTGATTAGTTCC-3’ (C1-J-1718 forward primer) 5’-CCGGATCCACNACRTARTANGTRTCRTG-3’ (H7005 reverse primer) ND5 was amplifi ed using primers as described by Yoshizawa (2004): 5’-ATCYTTWGAATAAAAYCCWG-3’ (ND5 F7081 forward primer) 5’-CCTGTWTCWDCTTTAGTWCA-3’ (R7495 reverse primer) All PCR 50-ul reactions consisted of: 1 ul template, 37.8 ul water, 5.0 ul 10× buffer with MgCl2 (Qiagen), 1.0 ul dNTPs (10mM, 50×), 2.5 ul of each primer (10 uM each), and 0.25 Taq (Qiagen). In all three cases, PCR reactions were initiated by a starting denaturation period of 2 min followed by 35 cycles of denaturation for 30 sec at 94° C and a fi nal elongation step of 7 min. Annealing temperature varied for each reaction: 40°C, 46°C, and 42°C for barcode, COI, and ND5, respectively, as did elongation temperature: 72°C, 72°C, and 65°C for barcode, COI, and ND5, respectively. DNA sequencing was carried out by Sequetech (Mountain View, California). Sequence alignment was done using ClustalW (Chenna et al. 2003) in MEGA supplemented by manual alignment.

We generated three trees. The fi rst tree used only the bar code sequence region that also incorporated sequences of taxa retrieved from BOLD, which allowed us to include more species of Ceuthophilus and Pristoceuthophilus than we were able to collect ourselves. A second tree was constructed using only our collected samples to fully utilize the 1273 bp we sequenced. Overlap of the two COI regions allowed us to concatenate these regions to create 860 continuous bp that matched positions 1552 to 2411 in the Drosophila yakuba Burla, 1954 mitochondrial genome (GenBank accession number X03240). ND5 sequences were concatenated to 858 bp (so that all bp were in the proper codon position) of the COI sequences and then analyzed. For the barcode sequences and the concatenated ND5 and COI sequences, phylogenetic trees were constructed using the neighbor joining (NJ) method using Maximum Composite Likelihood in MEGA with GTR + gamma substitution model (Tamura et al. 2004). A time tree using the GTR + gamma substitution model and an overall mitochondrial mutation rate of 0.023 substitutions per site per million years as described in Kaya et al. (2013) and Knowlton et al. (1993) was generated using only COI sequences using the algorithms in BEAST2 rather than NJ in MEGA (Drummond & Bouckaert 2015). Kaya et al. (2013) investigated the divergence times of various species of Dolichopoda (Bolivar, 1878) and Troglophilus (Krauss, 1879) (Orthoptera, Rhaphidophoridae) in

Table 1. Specimens used for phylogenetic analyses. The Canadian specimens are the Bold Systems bar code specimens. The specimen ID for these is the BOLD process ID. Specimen M8 is Pristoceuthophilus marmoratus Rehn, 1903.

Species/Sample Location Specimen ID Gryllus sp. Humboldt Co. California M9 Tropidischia xanthostoma Humboldt Co. California J3 (Scudder, 1861) Pristoceuthophilus A Los Angeles Co. California M8 P. B Lane Co. Oregon M5 P. C Humboldt Co. California M6 P. D Mariposa Co. J8 P. E Monterey Co. California M1 P. F Lane Co. Oregon M7 P. G Santa Clara Co. M16 P. H Mariposa Co. J7 P. I Santa Clara Co. M20 P. J Santa Cruz Co. M13 P. celatus (Scudder, 1894) Pacifi c Rim National Park British Columbia BBOWC278-11 P. cercalis (Caudell, 1916) Glacier National Park British Columbia BBOWC204-11 P. cercalis Hendrix Lake British Columbia INRMA2023-14 Ceuthophilus agassizii Kootenay NP British Columbia BBOWC082-10 C. brevipes Newfoundland Labrador SIOCN117-10 C. sp. Point Pelee National Park Ontario BBOWC280-11 Hemiudeopsylla californianus Santa Clara Co. Los Osos J5 H. californianus Mariposa Co. California J6 Rhachocnemis validus San Luis Obispo Co. California M11 Farallonophilus cavernicolus San Francisco Co. California M3

the context of the tectonics of the Eastern Mediterranean using COI sequences. Their results were generally consistent with tectonic events such as the opening of the Mid- Aegean Trench 9–12 mya. As a check on our BEAST methodology we constructed a time tree in BEAST using their publicly available COI sequence data and found results consistent with theirs. Concerns have recently been raised about nuclear mitochondrial pseudogenes (numts) and their possible confounding impact on phylogenetic studies for Orthoptera in general, and specifi cally the mtDNA regions we have used for this study (Song et al. 2008; Song et al. 2014). We have dealt with these issues in two ways. We followed the best practices outline for avoiding numt amplifi cation in mtDNA analysis (Song et al. 2008). For example, we knew a priori that all of the specimens collected were correctly identifi ed based on morphological characters or information from GenBank records. We only used sequences with clear bands and no chromatogram noise or ambiguity. Secondly, we validated that the resulting sequences were not those of pseudogenes. We checked for the non-occurrence of stop codons in our sequences. Also, a recent paper on the barcode region of the COI gene (Pentinsaari et al. 2016) points out that in the amino acid sequence of the barcode region of the COI gene there are 23 amino acid positions at which the residues are the same across the whole animal tree of life. The presence of variations in these positions would be an indication, like the presence of

Figure 1. Neighbor joining tree as computed by MEGA using Maximum Composite Likelihood and GTR substitution model with a gamma parameter of 1.2. Bold Systems sequences from Gen- Bank included. Sequences consist of 622 base pair barcode sequences. Despite the shorter sequences, the divisions between Farallonophilus and the Ceuthophilus and Pristoceuthophilus species remain the same. There is some rearrangement among the Pristoceuthophilus but lower level groups remain the same. Bootstrap values shown.

a stop codon, that we were dealing with a pseudogene. We found no such variations in our sequences. Moreover, the relationship outcomes are generally congruent with currently accepted taxonomic classifi cation (Cigliano et al. 2018). All sequences were entered in GenBank.

RESULTS AND DISCUSSION The phylogenetic relationship of F. cavernicolus to Pristoceuthophilini and Ceuthophilini is consistent between the three trees: COI barcode (Figure 1), COI plus ND5 concatenated (Figure 2), and the COI time tree (Figure 3). Our main result is that F. cavernicolus is not a member of Pristoceuthophilini, but instead diverged from both Pristoceuthophilini and Ceuthophilini roughly 1.6 million years before those lineages diverged from each other (Figure 3). All trees clearly show the division between two clades: one consisting of taxa identifi ed to the genus Pristoceuthophilus and another consisting of species currently placed in the Ceuthophilini: Ceuthophilus brevipes (Scudder, 1863), C. agassizii (Scudder, 1861), R. validus, and H. californianus. These clades form a monophyletic group, individually and together. This is supported both by the barcode tree (Figure 1), which contains more species, and the COI plus ND5 tree that uses longer sequences. The time tree is also consistent with these. The

Figure 2. Neighbor joining tree as computed by MEGA using Maximum Composite Likelihood and GTR substitution model all codon positions with a gamma parameter of 1.2. The sequences used were concatenations of 858 base pairs of our COI sequences with 384 base pairs of our ND5 sequences, chosen so that there are no partial codons. Note that the Ceuthophilus and Pristoceutho- philus species correctly separate into two groups and that Farallonophilus is an outgroup to both. Bootstrap values shown.

Figure 3. Time tree as computed by BEAST2 using GTR + GAMMA + I substitution model Times shown are millions of years. Only the COI sequences were used for this as the calibrations we used were based on COI. Times are based on a .023 per million years per site divergence rate.

main variation among the trees is the precise placement of Pristoceuthophilus J with regards to the other Pristoceuthophilus taxa. The only exception to the standard classifi cation as specifi ed in the Orthoptera Species File (Cigliano et al. 2018) is the placement of F. cavernicolus outside of these groups. The time tree using 858 bp of the COI region indicates that the divergence between F. cavernicolus and the Pristoceuthophilini/Ceuthophilini clade occurred roughly 8.9 mya, while the two tribes diverged more recently at roughly 7.3 mya. Subgroups of Ceuthophilini and Pristoceuthophilini diverged from each other in the range of 2–6 mya. It is suggestive that all these divergences starting roughly 9 mya coincide with a period of intense geological change in the near California region (Harden 2004). Processes such as the rise of the Sierra Nevada beginning about 9 mya and the elevation of many mountain ranges like the Coast Ranges 3.5–5 mya due to compression along the boundary between the North American and Pacifi c tectonic plates drastically changed topography and drainage patterns and created many opportunities for reproductive isolation and speciation. Of 11 Pristoceuthophilus species, six are found only in the near California region (one in Arizona, fi ve in California), according to the distribution maps in the Orthoptera Species File. The others occur in Washington and Mexico. The Ceuthophilini also have a strong presence in California, although they have a much broader distribution across North and Central America. Of course, F. cavernicolus is only found in California, and its nearest relatives have a strong presence in California. Therefore, it seems reasonable to hypothesize that the speciation events that created the Farallonophilus, Pristoceuthophilini, and Ceuthophilini lineages took place in the near California region and were related to geologic changes in that region. It has been suggested that the COI region that we

used for dating has a faster divergence rate than what we used (Papadopolou 2010). However, this would only compress the time scale of speciation events in our time tree without changing the ordering.

ACKNOWLEDGMENTS Michael Valainis thanks Pete Warzybok, Jim Tietz, and Ryan Berger of Point Blue for assistance in maintaining residence on the island. We also thank the U.S. Fish and Wildlife service for allowing us access to the island, and the Farallon Patrol for transportation to and from the island. We thank Professor David A. Gray of California State University, Northridge for a specimen of Pristoceuthophilus marmoratus. This work was supported in part by The Elinor Patterson Baker Trust, Giles W. and Elise G. Mead Foundation, Kimball Foundation, the Volgenau Foundation, and other individual Point Blue individual donors. This is Point Blue Contribution Number 2126.

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