Research Article |
Corresponding author: Kojun Kanda ( kk933@nau.edu ) Academic editor: Mariano Michat
© 2016 Kojun Kanda, R. Antonio Gomez, Richard Van Driesche, Kelly B. Miller, David R. Maddison.
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:
Kanda K, Gomez AR, Van Driesche R, Miller KB, Maddison DR (2016) Phylogenetic placement of the Pacific Northwest subterranean endemic diving beetle Stygoporus oregonensis Larson & LaBonte (Dytiscidae, Hydroporinae). ZooKeys 632: 75-91. https://doi.org/10.3897/zookeys.632.9866
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Stygoporus oregonensis Larson & LaBonte is a little-known subterranean diving beetle, which, until recently, had not been collected since the type series was taken from a shallow well in western Oregon, USA, in 1984. Here we report the discovery of additional specimens collected from a nearby well in the Willamette Valley. Sequence data from four mitochondrial genes, wingless, and histone III place Stygoporus Larson & LaBonte in the predominantly Mediterranean subtribe Siettitiina of the Hydroporini. Morphological support for these results is discussed, and details of the collecting circumstances of the new specimens are presented. We argue that the biogeographic patterns of Nearctic Siettitiina highlight the likelihood of additional undiscovered subterranean dytiscids in North America.
Stygobiont, aquatic Coleoptera, Hydroporini , aquifer, Siettitiina , Nearctic, Oregon
In the spring of 1984 an unusual, pale, blind diving beetle was found in a bathtub in a private residence near the town of Dallas, Oregon, USA. The bathtub received water directly from a shallow well that was drawing from the Willamette Lowland aquifer system in the central Willamette Valley. The residents sent the specimen to an entomology extension specialist, Dr. J. Capizzi at Oregon State University, who recognized the beetle as distinct and suggested to the residents that they collect more specimens (
Stygoporus oregonensis is a small-bodied diving beetle with pale, mostly yellow cuticle, long elytral marginal setae, fused elytra, minute flight wings, and without eyes (
Inferring the phylogenetic placement of stygobitic species is crucial for shedding light on their origins and developing a framework for studying adaptation and other responses to subterranean environments. Addressing the mechanisms responsible for the unusual though oft-repeated appearance of the stygobitic fauna and their often unexpected distributions is an active field (
In the United States, three aquatic beetle families are known to include stygobitic species: Dryopidae (Stygoparnus comalensis Barr & Spangler, 1992), Elmidae (Typhloelmis Barr, 2015: 3 species), and Dytiscidae (Ereboporus naturaconservatus Miller, Gibson & Alarie, 2009, Haideoporus texanus Young & Longley, 1976, Psychopomporus felipi Jean, Telles & Miller, 2012, Comaldessus stygius Spangler & Barr, 1995, and Stygoporus oregonensis Larson & LaBonte, 1994). Apart from S. oregonensis, all US stygobitic beetles are only known to occur in the Edwards-Trinity aquifer system in central Texas.
Whereas the relationships of Stygoparnus Barr and Spangler and Typhloelmis to other members of their respective families have yet to be explored with phylogenetic methods, the placement of three of the four described Texas stygobitic dytiscids within the very diverse subfamily Hydroporinae was recently inferred using molecular sequences (
In their paper describing S. oregonensis,
In this paper, we report additional specimens of S. oregonensis from a separate well, also in the central Willamette Valley, Oregon. These specimens yielded DNA, from which we amplified six genes used in
Two mostly intact specimens of Stygoporus oregonensis and fragments of additional individuals were recovered from accumulated sand and detritus in the filter of a residential well system (USA: Oregon: Marion County, Talbot, south of Talbot Road South). The well sits near an old oxbow of the Willamette River and the wellhead is located roughly 14 m below the surface. This site is roughly 27km SSE of the type locality (Fig.
The two known collection localities of Stygoporus oregonensis. Oregon/Washington State boundary in black. County boundaries in brown. Blue shaded region outlined with a dotted line corresponds to Willamette Lowland basin-fill aquifers. Type locality indicated by red star with black border. New collection locality indicated by black star.
The mostly intact beetle specimens were both caught during the rainy winter months and contained soft tissue, which appeared to be suitable for DNA extraction and PCR sequencing. The specimens were found with the prothorax and head slightly separated from the rest of the body and the genitalia extruded as if they had expanded slightly. This damage may have occurred during depressurization: the removal of the filter causes a change in pressure from 8-10 psi to atmospheric pressure in approximately 2 seconds.
In addition to S. oregonensis, we recovered crustaceans (ostracods, copepods, and Bathynellacea), numerous oribatid mites, and a few other insects (Throscidae (Coleoptera), Chironomidae larvae (Diptera), and unattributed elytral fragments). While the Throscidae appears to be an obvious terrestrial contaminant, we could not determine if the other taxa are associated with the aquifer or not. The pair of pale elytra recovered in one of the samples (
We extracted DNA from the two fairly intact specimens of S. oregonensis using DNeasy Blood and Tissue kits (Qiagen) following the manufacturer's protocols. Specimens were disarticulated between the abdomen and thorax prior to extraction; we did not grind any tissue, and thus the exoskeleton was preserved. We successfully amplified and sequenced six of the seven gene fragments used in
Initial assembly of chromatograms was performed using Phred v. 0.020425.c (
12S and 16S matrices were aligned using MAFFT v. 7.130b (
Optimal data partition schemes and model of molecular evolution for protein-coding genes were inferred using PartitionFinder v. 1.1.1 (
Properties of phylogenetic datasets analyzed for this study. NTaxa: The number of taxa represented in the dataset. Partitions: Optimal partitioning scheme chosen by PartitionFinder. NChar (BP): Number of characters (bases) in the aligned dataset/partition. Model: Optimal model of molecular evolution inferred by either jModelTest (12S, and 16S) or PartitionFinder (protein-coding genes).
Dataset | NTaxa | Partitions | NChar (BP) | Model |
---|---|---|---|---|
12S | 49 | NA | 362 | GTR+I+G |
16S | 50 | NA | 533 | HKY+I+G |
COI | 44 | (1) n1, n2 | 838 | GTR+I+G |
(2) n3 | 418 | GTR+G | ||
COII | 43 | (1) n1, n2 | 450 | GTR+I+G |
(2) n3 | 224 | GTR+G | ||
H3 | 50 | (1) n1, n2, n3 | 328 | GTR+I+G |
wg | 20 | (1) n1, n2 | 306 | GTR+I+G |
(2) n3 | 154 | GTR+G | ||
Concatenated | 51 | (1) 12S, 16S | 895 | GTR+I+G |
(2) n1 and n2 of all genes | 1812 | GTR+I+G | ||
(3) n3 of COI and COII | 642 | GTR+G |
We conducted Maximum Likelihood (ML) analyses on single gene and concatenated datasets using RAxML v. 8.0.3 (
Methods for gross morphological examination and use of terms follow
The dorsal habitus image was taken with a Leica Z6 and JVC KY-F75U camera using Microvision’s Cartographer to take a stack of pictures at different focal planes. Stacking was performed using the PMax procedure implemented in Zerene Stacker (Zerene Systems). Removal of background and minor color adjustment was performed using Photoshop and Illustrator CS5 (Adobe).
All specimens examined in this study and the two DNA extractions are deposited in the Oregon State Arthropod Collection (
Morphological characters discussed below are based on the original description of S. oregonensis (
The proventriculus of S. oregonensis has a simple transverse tooth similar to that of Hydroporus Clairville with fields of papillae laterally. The female genitalia are of hydroporine-type (
The maximum likelihood (ML) tree of the concatenated dataset is shown in Figure
Maximum likelihood tree from concatenated dataset. Scale bar = 0.2 expected substitutions per position as estimated by RAxML. Stygoporus oregonensis in orange; other stygobitic dytiscids in blue; the epigean genus Sanfilippodytes, hypothesized by
Bootstrap support for placement of Stygoporus oregonensis. Taxonomic hypotheses are in the first column. Bootstrap support given as a percentage for each hypothesis for all analyzed matrices. “Con” refers to the analysis of the concatenated matrix.
Taxonomic hypotheses | Con | 12S | 16S | COI | COII | H3 | wg |
---|---|---|---|---|---|---|---|
Stygoporus oregonensis + Ereboporus naturaconservatus | 99.3 | 79.2 | 41.1 | 60.1 | 70.7 | 86.0 | 90.0 |
S. oregonensis + Sanfilippodytes | 0 | 0 | 0 | 0 | 0.2 | 3.0 | 0.5 |
Siettitiina including S. oregonensis | 75.5 | 4.6 | 87.7 | 45.7 | 0 | 0 | 31.0 |
Siettitiina excluding S. oregonensis | 0 | 0 | 1.3 | 1.2 | 0 | 0 | 0 |
S. oregonensis in Hydroporina | 0 | 0.1 | 0 | 0 | 0 | 0 | 0 |
Maximum likelihood analysis of the concatenated dataset recovers S. oregonensis as sister to the Texas stygobite E. naturaconservatus (Figs
Ereboporus naturaconservatus and S. oregonensis are recovered as sister species in all single gene ML analyses (Suppl. material
In their original description of Stygoporus,
There are additional morphological characters in support of inclusion of S. oregonensis within Siettitiina, though it remains unclear whether these characters are strong synapomorphies for Siettitiina as a whole. In particular, the pronotum of S. oregonensis has prominent paralateral longitudinal creases or striae similar to many members of the larger group (e.g. Graptodytes Seidlitz, Siettitia Abeille de Perrin and Etruscodytes Mazza, Cianferoni, and Rocchi). The prosternal process of S. oregonensis contacts the anteriorly projecting and narrowly rounded metaventral process, resting dorsad to it and altogether looks remarkably similar to the Italian stygobite Etruscodytes. This region of the body has received much attention from biologists interested in stygobitic beetles (
Other morphological features in S. oregonensis relevant to grouping within Hydroporini are known plesiomorphies. These are, for example, the simple transverse tooth of the proventriculus, the unfused, simple gonocoxae, the basally broad and apically narrowed elytral epipleuron, the male pro- and mesotarsomeres I-III with ventral adhesive setae, and the mesoventral fork separated from the anteromedial metaventral process. Most of these characters are unlike those observed in Deronectina and Sternopriscina, and the morphological evidence separating Hydroporina from Siettitiina is limited. Based on our observations, it appears that Stygoporus retains many plesiomorphies and placement based on morphological characters alone is difficult. However, the sequence data support the inclusion of Stygoporus within Siettitiina, and they decisively indicate that Stygoporus is closely related to Ereboporus among sampled species.
The Siettitiina has a predominantly Mediterranean and European distribution and includes many epigean species as well as other subterranean species (e.g.
We sincerely thank the private landowners who allowed us access to their land. Portions of this project were supported by the Harold E. and Leona M. Rice Endowment Fund at Oregon State University (to D.R. Maddison) and NSF grants #DEB-0845984 and #DEB–1353426 (to K.B. Miller).
Figure 1
Data type: Adobe PDF file
Explanation note: Maximum likelihood trees for single gene datasets. Scale bar indicates the expected substitutions per site as estimated by RAxML.
Figure 2
Data type: Adobe PDF file
Explanation note: Majority rule consensus of 1,000 bootstrap replicates performed on single gene datasets. Bootstrap percentage given for clades recovered with more than 50% support.
Table 1
Data type: MS Word file
Explanation note: Collection and specimen data for material examined in this study.
Table 2
Data type: MS Word file
Explanation note: PCR primers and amplification conditions for sampled gene fragments.
Table 3
Data type: MS Word file
Explanation note: Taxa from
Data availability
Data type: NEXUS file
Explanation note: NEXUS formatted single-gene and concatenated nucleotide sequence alignments.