A peer-reviewed open-access journal ZooKeys | 106: 141—163 (2022) Eau ae tea #ZooKeys https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research Phylogenetic review of the millipede genus Cherokia Chamberlin, 1949 (Polydesmida, Xystodesmidae) Luisa Fernanda Vasquez-Valverde', Paul E. Marek' | Virginia Tech, Department of Entomology, 170 Drillfield Drive, Blacksburg, Virginia 24061, USA Corresponding author: Luisa Fernanda Vasquez-Valverde (luisafvv@vt.edu) Academic editor: Dragan Antié | Received 28 January 2022 | Accepted 5 May 2022 | Published 20 June 2022 http://zoobank. ore/856382C3-0061-465D-899A-704EF7797CF9 Citation: Vasquez-Valverde LE, Marek PE (2022) Phylogenetic review of the millipede genus Cherokia Chamberlin, 1949 (Polydesmida, Xystodesmidae). ZooKeys 1106: 141-163. https://doi.org/10.3897/zookeys.1106.81386 Abstract The millipede genus Cherokia Chamberlin, 1949 is a monospecific taxon, with the type species Cherokia georgiana (Bollman, 1889). The last revision of the genus was made by Hoffman (1960) where he estab- lished three subspecies. Here we used molecular phylogenetics to assess the genus and evaluate whether it is a monophyletic group, and if the subspecies are each monophyletic. We included material from literature records and three natural history collections. Newly collected samples were obtained through a citizen science project. Morphological characters underlying subspecies groups—the shape of the paranota, body size, and coloration—were evaluated. A molecular phylogeny of the genus was estimated based on DNA sequences for seven gene loci, and a species delimitation analysis was used to evaluate the status of the subspecies. The documented geographical range of Cherokia in the United States was expanded to include a newly reported state record (Virginia) and about 160 new localities compared to the previously known range. Morphological characters, which included the shape of the paranota and body size that had been historically used to establish subspecies, showed clinal variation with a direct relationship with geographi- cal distribution and elevation, but not with phylogeny. Coloration was highly variable and did not accord with geography or phylogeny. The phylogeny recovered Cherokia as a monophyletic lineage, and the spe- cies delimitation test supported the existence of a single species. The subspecies Cherokia georgiana ducilla (Chamberlin, 1939) and Cherokia georgiana latassa Hoffman, 1960 have been synonymized with Cherokia georgiana. The molecular and morphological evidence showed that Cherokia is a monospecific genus with the sole species, Cherokia georgiana, being geographically widespread and highly variable in its morphology. Keywords Citizen science, DNA barcoding, morphology, phylogenetics, subspecies Copyright L. Fernanda Vasquez-Valverde & Paul E. Marek. 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. 142 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) Introduction The family Xystodesmidae (Polydesmida) includes 539 species with a center of diver- sity concentrated in the Appalachian Mountains (Means et al. 2021a, b; Hennen et al. 2022). Within the family Xystodesmidae, the Appalachian genus Cherokia Cham- berlin, 1949 (Fig. 1) was named after the Cherokee, an indigenous group of peoples in the southeastern United States. This monospecific genus in the xystodesmid tribe Rhysodesmini was erected by Chamberlin (1949) for the species Fontaria georgiana Bollman, 1889 as its type species. After its description, various authors proposed mul- tiple species that were all subsequently synonymized with the type species Cherokia georgiana (Bollman, 1889) based on gonopod morphology (Causey 1950; Hoffman 1950; Chamberlin and Hoffman 1958). However, all the above-mentioned authors pointed out the considerable color, body size, and shape variation in millipedes of the genus Cherokia. Prior to Hoffman's (1960) revision, no one had carried out a comprehensive synthesis of this genus. He (Hoffman 1960) proposed Cherokia as a monospecific genus, with the sole species Cherokia georgiana divided into three subspecies: Cherokia georgiana georgiana (Bollman, 1889), Cherokia georgiana ducilla (Chamberlin, 1939), and Cherokia georgiana latassa Hoffman, 1960. Hoffman (1960) described in detail ~ = <= 2 am, — “<. Figure |. Cherokia georgiana (Bollman, 1889), the wrinkled flat-backed millipede. Dorsal view of the whole body of specimen MPE04539 (Male, GA — White Co.) deposited in the Virginia Tech Insect Collection (VTEC). The image shows the more common coloration for the species and the prominent wrinkles of the cuticle. Phylogenetic review of the millipede genus Cherokia 143 the morphological variation and geographical distribution of Cherokia. He also differentiated the three subspecies from each other based on morphological features that included the position of the scapulora and the ratio of the body length versus its width. The scapulora is a term defined by Hoffman (1960: 231) as: “from the Latin “scapula,” a shoulder, and “ora,” the rim of a shield”. The scapulora in C. g. latassa is found in a marginal position, which separates it from C. g. georgiana and C. g. ducilla that have a submarginally located scapulora (Fig. 2A, B). The subspecies, C. g. georgiana and C. g. ducilla, were differentiated from each other based on the ratio of the previously mentioned body measurements (i.e., body length versus its width) (Hoffman 1960). Hoffman (1960) confronted various problems during his revision of the genus Cherokia. The first one, he explained, was the fact that “despite the diversity of body form, color pattern, and morphological details which occurs in the genus, the male gonopods remain essentially similar” (Hoffman 1960: 227). Although some variation in the solenomere shape in specimens in the North Carolina mountains was observed by Hoftman (1960), the character was not consistent and did not correlate with geo- graphical distribution or subspecies differentiation. Additionally, the same author ex- pressed a struggle with confidently assigning all individuals to one of the subspecies. For this reason, Hoffman (1960) proposed an intermediate form, termed an “inter- grade” between C. g. georgiana and C. g. ducilla. These intergrades were documented within a wide geographical band (~30 km) in western North Carolina between the distributions of C. g. georgiana and C. g. ducilla. A B Sc Sc tae Wa Figure 2. Position of the scapulorae (Sc) A strictly marginal B submarginal. Measurements of the 12" body ring C metazonite width D metazonite length E paranota extension. Adapted from Hoffman (1960). 144 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) After 1960, some authors have indirectly mentioned Cherokia in tribal revisions (Hoffman 1978) and checklists (Shelley 1980, 2000; Marek et al. 2014). More recent research on the family Xystodesmidae, using a synthesis of morphological and mo- lecular characteristics, has confidently placed the genus Cherokia within the family Xystodesmidae and subfamily Rhysodesminae Brolemann, 1916 as sister to the genus Pleuroloma Rafinesque, 1820 (Means and Marek 2017; Means et al. 2021b). As a result of field collections for this recent work, a large number of Cherokia individuals were collected from throughout the eastern United States, and within its range. These recent results combined with materials from natural history collections from the mid-1900s up to now, point to an even greater diversity than initially uncovered. Here we used natural history collections in combination with new material sam- pled from nearly 200 locations within the range of Cherokia. These new samples, spe- cially prepared for preservation of DNA, provided the basis to estimate an evolutionary history using molecular phylogenetics and address the status of the three subspecies within Cherokia georgiana. Materials and methods Selection of samples and Citizen science project Specimens of the genus Cherokia preserved in the Virginia Tech Insect Collection were selected based on the availability to score both morphological and molecular characters from them. Individual live millipede specimens or their tissues were fixed in either 100% ethanol or Qiagen RNAlater thereby preserving DNA and other genetic mate- rial. Whole body specimens (minus tissue preserved for DNA) were then preserved in 70% isopropanol for subsequent morphological evaluation. New samples were needed from some localities that had not previously been sam- pled; these localities were in the periphery of the known distribution of Cherokia or in areas where DNA-grade specimens were unavailable. A season of fieldwork was planned for the Summer 2020, however, due to the SARS-CoV-2/COVID-19 pan- demic, and state restrictions, travel was not feasible. In response, and with the objec- tive of obtaining these required samples, a citizen science project was developed. ‘This enabled the general public to participate in the collection of millipedes of the genus Cherokia, and to learn about biodiversity. For the citizen science project, collection kits and information pamphlets were de- signed with step-by-step instructions and other information for the public to obtain samples in an accurate and lawful way (Fig. 3B). Citizen scientists were recruited with social media through Facebook and Twitter, and the kits were shipped to interested par- ticipants. A small plastic keychain of Cherokia was included in the kit as a token of par- ticipation (Fig. 3E). Once the participants received the kit and collected millipedes, they were instructed to ship the millipedes to the lab at Virginia Tech, so we could identify, process and preserve them following methodology described by Means et al. (2015). Phylogenetic review of the millipede genus Cherokia 145 TU Cherokia Collection Citizen Science Project ENTOMOLOGY SYSTEMATIC LAB Figure 3. Citizen science collection kit A plastic food container (32 FL OZ) B instruction flyer: with step-by-step instructions of collecting and shipping C clear plastic collection vials D collection card E token for the participant: millipede keychain and F Cherokia identification card. DNA extraction, phylogenetics analysis and species delimitation Preserved tissue (legs or head) from each individual was used for DNA extraction with a Qiagen DNeasy kit. The DNA obtained from the extraction was amplified via polymerase chain reaction (PCR) for seven gene regions: cytochrome oxidase subunit I (COI), small subunit RNA (12S), tRNA-Valine (tRNA-Val), large subunit RNA (16S), elongation factor-alpha (EFla«), RNA polymerase II largest subunit (RNAPol2), and F-box (fBox). The mitochondrial 12S, tRNA-Val, and 16S regions were amplified as a single contiguous stretch. Amplification of DNA was carried out according to Means et al. (2021a, b). These PCR amplicons were cleaned, quantified, normalized, and sequenced on an Applied Biosystems ABI 3730 capillary sequencer at the Univer- sity of Arizona Genetics Core. The sequences were analyzed in Mesquite (Version 3.61) (Maddison and Maddison 2019) using the sequence analysis module Chromaseg (Version 1.52) that implements phred and phrap (Ewing et al. 1998; Maddison and Maddison 2020) for chromatogram base calling, trimming, quality control and generation and curation of matrices. The outgroups were selected based on the phylogeny inferred by Means et al. (2021b) and included a single individual of: Pleuroloma flavipes Rafinesque, 1820, Pleuroloma plana Shelley, 1980 and Pleuroloma cala (Chamberlin, 1939). Sequences were aligned with the progressive sequence alignment program MAFFT (Version 7) using the model L-INS-I (Katoh and Standley 2013). A nucleotide base composition homogeneity chi-square test in IQ-TREE 2 (Version 2.0.4; Minh et al. 2020) was run with the aligned sequences for each of the genes to test the heterogeneity of the sequences (H = homogeneity) and excluding the sequences of the outgroup taxa. The sequences that failed the heterogeneity test were excluded from the phylogeny. alternative Afterwards, each locus was partitioned by gene, intron/exon location, and codon y 8 position. The seven loci were concatenated into a single matrix. The partitioned matrix 146 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) was analyzed using ModelFinder to test alternative nucleotide evolution models and to infer the model of best-fit for the data (Kalyaanamoorthy et al. 2017). The selected model was then used to estimate a phylogenetic tree for the genus with the maximum likelihood-based phylogenetics software IQ-TREE 2. Single gene alignments were then analyzed separately to estimate gene trees with the same methods and software as above. To determine whether the subspecies of Cherokia georgiana represent distinct ge- netic groups, Automatic Barcode Gap Discovery (ABGD) species delimitation analysis was used. This method uses an alignment of sequences of a single locus (COI) to make a pairwise distance matrix and determine if a barcode gap exists. A barcode gap is observed when the intraspecific distance among unique sequences is smaller than the interspecific distance (Puillandre et al. 2012). This analysis was run in the ABGD online server using the alignment of Cherokia sequences for the locus COI, excluding the outgroup sequences. Distribution mapping and morphological characters analysis To infer a detailed geographical range of the genus Cherokia, records in the literature, natural history collections, and new collections from the citizen science project were included. All the localities of specimens of Cherokia documented in Hoffman (1960) and from the Virginia Tech Insect Collection, Virginia Museum of Natural History (VMNH), and Florida State Collection of Arthropods (FSCA) were digitized. Digi- tization involved transcribing the label data of specimens in a spreadsheet using the Darwin Core data standard (Wieczorek et al. 2012). Text-based details of the label including state, county, and any other locality information were manually entered in a spreadsheet. In cases where precise geographical coordinates (e.g., latitude and lon- gitude) were not provided, the text of the localities from the labels was transcribed, georeferenced and geographical coordinates automatically estimated using the software GEOLocate (Rios and Bart 2010) to retrospectively obtain decimal degree coordinates and an error radius based on precision of the text locality. To supplement this data set, localities from Cherokia specimens from the Virginia Tech Insect Collection (VTEC) that were already digitized and with geographical coordinates recorded at the time of collection were downloaded from the online database SCAN (Barkworth et al. 2019). This data set of coordinates (from collections and literature), was the basis to produce a comprehensive map of the geographical range of Cherokia. The map was constructed in the online GIS application SimpleMappr (Shorthouse 2010). For the analysis of morphological features, the traits described in Hoffman (1960) were revisited: width-to-length ratio, color (hue and pattern), gonopods, and the posi- tion of the scapulora (Fig. 2A, B). Hoffman (1960) measured the entire length of the trunk of the millipedes; however, due to the flexibility of the trunk and the rings that make up the trunk—causing accordion-like compression and extension—these overall length measurements typically have a large error. With the idea of evaluating size vari- ation more accurately, the 12" body ring only was dissected from each specimen and measured for: (1) width (Fig. 2C) and (2) length (Fig. 2D) of the metazonite in dorsal Phylogenetic review of the millipede genus Cherokia 147 view; and (3) the paranota lateral extension from a posterior view (Fig. 2E). Measure- ment of a single ring reduces the error, because a single diplosegmental ring is rigid and inflexible, and hypothetically linearly correlated to overall length. To control for a non- normal body size distribution, a natural logarithm to transform the raw measurements was used. Linear regressions were then used to evaluate potential correlations between the measurements and elevation. Cherokia georgiana exhibits a considerable diversity in coloration patterns through- out its geographical distribution. To evaluate this variation, pictures of the species taken from the specimens selected for the analysis and those observed on iNaturalist (available from https://www.inaturalist.org; accessed May, 2020) were included. Be- fore including pictures from iNaturalist, identifications of the observations of Cherokia were confirmed by the authors (accessed on 18 May 2020). Afterwards the pictures were coded based on selection of one of three hue (red, orange, and yellow) and pattern groups (bimaculate, trimaculate, and striped), and scored. These pattern codes were then mapped onto the distribution of Cherokia to test if there is a correspondence with geographical areas and phylogenetic relationships. Results Selection of samples and Citizen science project The citizen science campaign on social media received more than 100 responses from a Facebook and Twitter post. This resulted in 68 people who completed a Google form expressing their interest to participate in the project. Fifty people were then selected based on their location and proximity to areas previously not surveyed. Due to the limited number of kits available, sampling efforts were focused on the collection of millipedes in targeted localities in Georgia, Alabama and Tennessee. A total of 41 kits (Fig. 3) were shipped between the months of July and August of 2020 to participants who provided all the required information in the online form. From October 2020 to March 2021, a total of 23 live millipedes were received as a result of this project, and 13 of them were identified as Cherokia and included in the morphological and molecular analysis. A total of 106 individuals from the genus Cherokia were included in the molecular phylogenetic analysis: 74 males, 31 females and one juvenile. Of these, 88% of the selected samples were previously deposited at the VTEC, and the remaining 12% cor- responded to new samples obtained from the citizen science project. DNA extraction, phylogenetics analysis and species delimitation The amplification and sequencing of DNA for the loci, COI, 12S, tRNA-Val and 16S, had a high rate of success, and only one specimen did not amplify (Suppl. material 1). For the locus fBox, the rate of success in amplification and sequencing was 96%, and for the 148 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) loci EFla and RNAPol2 that rate was considerably lower with 75% and 55% of the total sequences obtained. When amplifications and/or sequencing failed, amplifications were repeated up to three times using the same DNA extraction before discontinuing attempts. The multiple sequence alignment in MAFFT and inference of nucleotide evolu- tion models in ModelFinder resulted in a 3865 bp concatenated matrix divided into six partitions and composed of 142 bp of 12S (TIM+F+G4 nucleotide evolution mod- el), 82 bp of tRNA-Val (TIM+F+G4), 1081 bp of 16S (TIM+F+G4), 600 bp of COI (posl TN+I+G4, pos 2’ TIM3+F+R2 and pos 3 TIM3+F+G4), 585 bp of EFla (pos 1 & 2 TN+1+G4, pos 3 TIM3+F+R2 and intron GTR+F+I+G4), 978 bp of RNAPol2 (pos 1, 2, 3 & intron 1 TN+F+R2 and intron 2 TIM+F+G4), and 397 bp of fBox (pos 1 & 2 TN+I+G4 and pos 3 TIM3+F+R2). Of the 3865 nucleotide characters, 2726 corresponded to constant sites, 738 were parsimony-informative, and 401 were singleton sites. The average uncorrected pairwise distance for COI sequences between individuals from the same locality was 0.00470 (max. = 0.01644, min. = 0, o = 0.005), and in total 0.07704 (max. = 0.12105, min. = 0, o = 0.02742). The maximum uncor- rected pairwise distance (COI) between Cherokia and Pleuroloma was 0.14740. The estimated phylogeny for Cherokia using the seven loci and the above-mentioned parti- tions and models is shown in Fig. 4. The ABGD analysis included high-quality COI sequences for 105 specimens of Cherokia and excluded the sequences from the outgroup taxon Pleuroloma. The analy- sis was carried out on the ABGD web server using the Jukes-Cantor (JC69) substitu- tion model and a relative gap width of 1.5X. The results of this analysis showed that a barcode gap does not exist in the COI sequences of Cherokia (Fig. 5A), and supports the model that all the individuals belong to the same group. Fig. 5B depicts what an expected histogram with a barcode gap present would look like; the dotted line marks the separation between the two groups and represents the likelihood of two species. Distribution mapping and morphological character analysis A total of 201 reports were digitized and georeferenced from Hoffman (1960) (N = 103), the VMNH (NV = 31) and FSCA (NV = 67) natural history collections. Local- ities from the VTEC were obtained (already databased), thereby adding 222 Cherokia records to the database. The map for the geographical distribution of the genus Cher- okia (Fig. 6) was constructed using 253 coordinates from localities representing 848 individuals. The geographical distribution includes seven states: Alabama, Georgia, Kentucky, North Carolina, South Carolina, Tennessee and Virginia. Ninety-six coun- ties from throughout the aforementioned states have records of Cherokia individuals. All of the adult individuals used for the phylogeny were included in the morpho- logical analysis. The juvenile (Fig. 4; GA-TIF-MPE03692) was excluded due to lack of development in its morphological characters, which could have introduced unwanted outliers and substantial error in the data set. The measurement from the metazonal width had the greatest variation range (range = 6.0-9.3 mm, o = 0.74, N = 105), fol- lowed by the paranotal extension (range = 1.25—2.17 mm, o = 0.24, N = 105), and lastly by the metazonal length (range = 1.54—2.60 mm, o = 0.20, NV = 105). 0.01 site substitutions 0.1 Phylogenetic review of the millipede genus Cherokia 100.0 $1.01 149 Pleuroloma cala Pleuroloma flavipes Pleuroloma plana AL-MAD-MPE01272 100.08 AL-MAD-MPE04662 AL-CAL-SPC001124 AL-JAC-SPCO00469 91.0) 76.0) 98.0) 100.0 30.9 100.0 80.0} 91.0 83.0) 90.0) AL-JAC-SPC000064 GA-FLO-MPE02360 4% AL-MAR-SPCO00060 39.0) AL-MAR-SPCOO0061 100.08 AL-MAR-SPCO000062 AL-LAW-SPC000975 AL-WIN-MPE01263 100.08 AL-WIN-MPE01336 TN-DIC-MPE02700 TOO. TN-DIC-MPE02683 71.0 TN-DIC-MPE02 702 AL-JAC-SPC000073 TN-WIL-SPC000477 98.0h TN-DAV-SPC000490 KY-HRL-SPCO000174 KY-WTY-SPCO00604 VA-LEE-MMC0264 TN-CUM-SPC000009 KY-PUL-MPE03215 KY-PUL-MPE03216 | VA-DCK-SPC000307 VA-LEE-MMC0265 KY -PUL-MPE03167 = VA-LEE-MMC0269 TN-CAM-MPE00540 KY-MCC-SPC000597 VA-WIS-MPE03992 TN-MOR-MPEO2181 KY-HRL-SPC000790 TN-MOR-MPE02823 KY-PIK-MPE03234 KY-KNO-MPE03252 TN-PIO-MPEO00535 TN-VBU-MPE01258 NC-HAY-MPE01547 NC-JAC-MPE01473 TN-COC-SPC001108 TN-SEV-MPE04820 O00 TN-SEV-MPE01508 100.0& TN-SEV-MPE04808 GA-LUM-SPC000050 100.0 92.0) 100.0} GA-TOW-MPE03316 GA-LUM-MPE04365 GA-LUM-MPE04345 GA-UNI-MPE04422 GA-DAW-MPE01308 GA-DAW-MPE01271 GA-RAB-MPE01822 GA-MUR-SPC000053 GA-MUR-SPC000054 GA-MUR-SPC000056 NC-GRA-MPE05013 NC-GRA-SPC000917 NC-YAN-MPE05014 NC-MAC-SPC000897 NC-MAC-SPC000903 SC-GRE-MPE01532 To0.0 SC-GRE-SPC000045 SC-OCO-MPE04551 i SC-OCO-MPE00514 80.se 0 SC-OCO-MPE04515 = SC-WAL-MPE00517 §C-OCO-MPE04550 SC-OCO-MPE04552 NC-MAC-MPE00506 96.0kes NC-HIG-MPE00512 NC-JAC-MPE00523 ' NC-HIG-MPE00490 0 i NC-HIG-MPE00493 3.08 NC-MAC-MPE00499 NC-MAC-MPE00501 NC-MAC-MPE01512 NC-MON-MPE05011 TN-MON-SPC000911 NC-GRA-SPC000892 NC-GRA-MPE05012 NC-GRA-SPC000035 NC-MAC-SPC000354 GA-RAB-MPE04383 GA-RAB-MPEO1585 T5-Okee GA -RAB-MPE04376 GA-TOW-MPE04356 GA-WHI-MPE04558 - GA-BAR-MPE03902 GA-JAC-MPE04252 72.0) 100.0 89.0 100.0 32.0) ; 52.0 95.0 00.0 93.0) GA-TOW-MPE04408 GA-WHI-MPE04539 GA-TOW-MPE03318 GA-UNI-MPE03331 GA-TIF-MPE03692 ++ GA-HAR-MPE05090 GA-LAM-MPE05057 GA-LAM-MPE05058 GA-LAM-MPE05059 AL-TAL-MPE05094 Too] AL-TAL-MPE05093 88.0h AL-TAL-MPE05095 AL-MAC-MPE05052 AL-MAC-MPE05088 gente AL-MAC-MPE0S071 92.0fy AL-MAC-MPE05064 AL-MAC-MPE05087 | I 0.0 92.0 100.0) 89.0 100.0) Figure 4. Phylogenetic reconstruction of the genus Cherokia Chamberlin, 1949. Terminals include the state, county and unique specimen code (i.e., AL-MAD-MPE01272). + Juvenile. * Outlier. 150 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) coo oc oO oO OO OC oO Ohl OUCUCOOUCUOUCUCUMUCUCOCUCUCUCOCUCOCUCCOUC Dist. value Soo So GO oO oOo ooo ol Go ol Glo Ol ol lol lo Dist. value C—O 2 SS SSC SCO CO CPP PPS SS SS Figure 5. ABGD analysis results A Cherokia Chamberlin, 1949 sequences, no barcode gap observed B simulated sequences, barcode gap marked by the dotted line. Once all the measurements were log-transformed, a linear regression analyzing the correlation between elevation and body dimensions were conducted for each of the respective measurements (Fig. 8). These analyses suggest that, in general, there is a negative correlation between the body measurements and the elevation; millipedes with smaller body sizes tended to be present in a higher elevation than those with a larger size. The position of the scapulora as described in Hoffman (1960) (Fig. 2) could not be consistently discerned and objectively scored and was not included in this analysis. Nev- ertheless, a qualitative difference in the shape of the anterior border of the paranota was observed and generally showed two phenotypes for this character. The first phenotypic group includes a distinct sinuous curvature on the anterior border of the paranota, while the posterior paranotal corner protrudes backwards posteriorly beyond the margin of the posteromedial margin of the metazonite (Fig. 9A—C, blue lines). The second phe- notypic group includes an almost straight anterior border, and the posterior corner is nearly aligned with the posteromedial margin of the metazonite (Fig. 9D, E, red lines). The coloration analysis of Cherokia included a total of 124 images of individuals identified as Cherokia on iNaturalist. The identifications of Cherokia observations on iNaturalist were confirmed by the authors based on the diagnosis below. The pictures were coded using the three colors (red, orange and yellow), and three patterns (bimacu- late, trimaculate, and striped). Most of the individuals exhibited only one of the colors, and a smaller proportion of them exhibited two. The color white was only observed present while in combination with another color (i.e., white and orange), while the other colors were present by themselves or with another. In the bimaculate pattern, a spot of color was present laterally on each paranota (there are two paranota per ring) with the center lacking pigmentation (Fig. 10A— C). The trimaculate pattern, is characterized by a coloration spot on each para- nota in addition to a middorsal spot on the ring. The middorsal or paranotal spots had different sizes and could be one of three shapes: a circle, oval, or a triangle (Fig. 1OD-—F). The striped pattern is where a color band is on the posterior margin of the body ring that runs from one paranota to the other. The band could have Phylogenetic review of the millipede genus Cherokia 151 Illinois iS e a West Virginia Eas J — Kentucky o eS tere irginia i ; ee oe? Je = ~™ — — = oa” @% @ pie e * ® e ; ® A. PS r ehh e e a a ies . S pete gs North Carolina el ee Ore == | Qype e e = —e | e °~ °| e e So ~ _ e \ a e NY PM i | ee *e. 'e ~\ South Carolina “F ef \ € | % | e Mississippi / Pisani \" e e e e e** Fate e a | . ; | | By \ r 4 | = = Flori : Bae. sk Figure 6. Geographical distribution of the genus Cherokia Chamberlin, 1949. Mapped using a set of 235 coordinates, from 848 individual records from Hoffman (1960), and natural history collections (VWMNH, FSCA, VTEC). various thicknesses, and in some cases an apparent superposition of the trimacu- late pattern was evident atop the banded pattern (Fig. 10G—I). There was no clear relationship between geographical distribution and the color or patterns; in some cases, syntopic individuals of Cherokia from the same locality exhibited different coloration patterns. Taxonomy Family Xystodesmidae Cook, 1895 Subfamily Rhysodesminae Brolemann, 1916 Tribe Rhysodesmini Brolemann, 1916 Genus Cherokia Chamberlin, 1949 Type species. Cherokia georgiana (Bollman, 1889) Cherokia georgiana (Bollman, 1889) Vernacular name: Wrinkled Flat-backed Millipede Fontaria georgiana Bollman, 1889a: 344. MALE HT (United States National Mu- seum, USNM). United States: Georgia, Bibb County. 152 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) Fontaria tallulah Bollman, 1889a: 344. FEMALE HT (USNM). United States: Geor- gia, Habersham County. Synonymized by Hoffman, 1950b: 23. Mimuloria furcifer Chamberlin, 1940a: 282, fig. 1. MALE HT (USNM). United States: North Carolina, Buncombe County. Synonymized by Hoffman, 1950b: 23. Mimuloria georgiana — Loomis 1943: 402. Dynoria parvior Chamberlin, 1947: 10, fig. 4. MALE HT (USNM). United States: Georgia, Union County. Synonymized by Hoffman, 1950b: 23. Cherokia georgiana — Chamberlin 1949a: 3. Cherokia georgiana georgiana Hoffman 1960: 240, figs 3d, 4e, 5a, 6, 7. syn. nov. Mimuloria ducilla Chamberlin, 1939: 7, fig. 12. MALE HT (USNM). United States: North Carolina, Jackson County. Mimuloria georgiana (nec Bollman, 1889) — sensu Loomis, 1943: 402. Cherokia georgiana ducilla Hoffman 1960: 255, figs 3b-e, 4f, 5b, 6, 7. syn. nov. Cherokia georgiana latassa Hoffman, 1960: 257, figs 3a, c, 4a—e, 5c, d, 7. MALE HT (USNM). United States: Tennessee, Warren County. syn. nov. Note. For a complete taxonomic listing, see Means et al. (2021b), Suppl. material 1. Diagnosis. Adults in the genus Cherokia are distinct from other rhysodesmine genera based on the following combination of characters: Body rings: dorsal surface of the metazonites with a noticeably wrinkly texture. Paranota horizontal and wide, with little downwards curvature, making the body appear flatter than other rhysodesmines. Gonopods: Telopodite sublinear in shape (Fig. 7), not distinctly curved or twisted as in the Apheloriini. Telopodite with a cingulum. Acropodite with its apex appearing flat and truncated. Telopodite with a long acicular prefemoral process; not a stout, curved prefemoral process nor wholly lacking as in the Apheloriini. Cyphopods: receptacle absent. Coloration: yellow to red hues in bimaculate, trimaculate and striped patterns (Fig. 10). Yellow trimaculate is the most frequent color morph (Fig. 1). Discussion The previously reported geographical range of Cherokia sensu Hoftman (1960), included six states, 43 counties and 93 localities. Here we report the presence of Cherokia in a seventh state (Virginia) and 53 new counties, for a total of 160 new localities where specimens of the genus have been collected. In prior systematic analyses of the millipede family Xystodesmidae, Cherokia was represented by three individuals, sequenced for six genes (Means et al. 2021b). Here, to address species boundaries in greater detail, we increased this number to 106 individuals sequenced for seven genes, for a total of 450 sequences and 3865 base pairs of DNA. These sequences were used to infer a phylogeny (Fig. 4). Of the seven loci amplified and sequenced for the phylogenetic reconstruction of the genus Cherokia, RNAPol2 was less successful than others in terms of amplification (presence of bands on electrophoretic gels) and sequencing (low quality reads: phred scores > 20). The presence of stop codons in RNAPol2 sequences, Phylogenetic review of the millipede genus Cherokia 153 despite viewing in six alternative reading frames, is unexpected and may indicate that it is a recent pseudogene. However, the relatively lower success in sequencing of this locus does not appear to affect the general topology of the phylogeny. Based on the molecular phylogeny, Cherokia is a monophyletic taxon (Fig. 4) sister to Pleuroloma. There is a clade formed by two individuals from the same locality (Monte Sano State Park, Madison Co., Alabama) that is sister to the remaining ones. Three statistically well-supported clades are present and subtended by long branches; however, the other individuals in the phylogeny are paraphyletic with respect to these clades and are not reciprocally monophyletic with them. In general, individuals from the same locality or nearby localities grouped together. Individuals from Kentucky and Virginia occur together with some individuals from Tennessee in a clade with very short branches. This block of individuals corresponds with the northeastern limit of the geographical range of the genus, and to the Cumberland Mountain Thrust Block region, a mountainous and complex region lying between the dissected Appalachian Figure 7. Scanning electron micrograph of a Cherokia georgiana male gonopod. Medial view of specimen MPE04252 (VTEC). 154 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) r R?=0.38 ; ° R?=0.31 Metazonal Width Metazonal Length 0 500 1000 1500 2000 Minimum Elevation (m) Minimum Elevation (m) ° R? = 0.43 Paranota Extension ' T T T T T T T - r y . - . 0 500 1000 1500 2000 1.8 19 2 4 292 Minimum Elevation (m) Figure 8. Linear regression of the elevation and body measurements A metazonal width B metazonal length C paranota extension and D Ln-transformed metazonal width distribution. Plateau to the west and the Valley and Ridges to the east. This region also houses a clade of millipedes in the genus Brachoria Chamberlin, 1939 with similarly very shallow genetic divergences as Cherokia (Marek 2010). These shallow branches in Cherokia, as in Brachoria (Marek 2010), may represent relatively more recent and/ or rapid diversification in the area, and may be due to shared mechanisms of regional diversity, or be associated with mimicry evolution in the area. Cherokia is a known participant of Miillerian mimicry in the region (Marek and Bond 2009; Marek 2010). The morphological characters evaluated by Hoffman (1960) were reexamined with new measurements and compared to geographical variables (i.e., elevation) and the phylogeny. The measurements taken from the 12" body ring and its inverse linear cor- relation with elevation showed that, in general, individuals of Cherokia with smaller body size and shorter paranota tend to be present at higher elevations than those with a larger size and longer paranota (Fig. 8). While the new measurements showed the same distribution as Hoffman (1960), the variation appears to be clinal, and not dis- cordant variation with abrupt changes that would be expected to correspond to species boundaries. Many terrestrial invertebrate taxa show smaller body sizes at higher eleva- Phylogenetic review of the millipede genus Cherokia 155 Sinuate paranota Straight paranota Figure 9. Variation in the paranota shape in Cherokia Chamberlin, 1949. Images of the 12th body ring of males (VTEC) showing sinuate paranota A SPC000060 (AL-MAR) B MPE01272 (AL-MAD) C MPE01336 (AL-WIN) or straight paranota D MPE02360 (GA-FLO) E MPE01308 (GA-DAW) F MPE01822 (GA-RAB). Blue and red lines denote the differences between the paranota shape. tions, but the converse has also been observed (Hodkinson 2005). Smaller Cherokia at higher elevations may be associated with resource limitation as has been implicated in other terrestrial invertebrate groups (Hodkinson 2005). Alternatively, the smaller body sizes may be associated with body shape differences linked to burrowing efficiency in different leaf-litter substrates at higher elevations (e.g., there is a greater diversity and abundance of evergreen trees at higher elevations). The results of the ABGD analysis showed a congruent pattern where genetic dis- tances are continuously distributed and no barcode gap exists (Fig. 5A). This shows that there are no clear genetic clusters indicative of a barcode gap for distinct species or subspecies (Fig. 5B). While sampling effort may affect ABGD analyses, our dataset of 106 specimens uniformly sampled from across the distribution of the genus supports the hypothesis of a single, widespread species. "WL QT S¥eq ALIS “DALLA ey} Woy are suaureds [TY “(NTH-ON) 10S00adW I (ODO -JS) SISPOAUW H (DIH-ON) SOSOOAdW © pedis pure “(QqgA-N.L) ZZZIOAMW A (AGA-NLL) SZTZIOAdUW A (ATS-NLL) 80STOAdW G 2ETHOeUID “QYOW -N.L) I8IZ70OAdW D alee, ZSTHOAMW @ (OVW-ON) ZISIOAIW W BENOeUTG “GHGT “UlPequieyD wz012q) Ul paAsrosqo sussiIed UONRIO[OD *Q] oundid i Taha cqucgenaaaa tet oe ig DS \ TS RBLERLELLLA eigen basses ets : ws Ar L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) ” DO DdDDP) & a Se * 156 Phylogenetic review of the millipede genus Cherokia 157 The position of the scapulora (sensu Hoffman 1960) was not a useful character, due to the difficulty of distinguishing its two states from each other (marginal and submarginal); perhaps this is due to its continuous nature, as is the case with the body size characters above (Fig. 8). During the examination of this character, we observed that the two states (marginal and submarginal) were not phylogenetically or geographically concordant. As described above (Fig. 9), the anterior margin of the paranota roughly grouped into two distinguishable shapes: sinuate or straight. To evaluate the relevance of this newly discovered character, its geographical distribution was mapped (Fig. 11). The geographical distribution shows that the individuals with sinuate paranota generally tend to be located in the western part of the Appalachian region, while the individuals with a straight paranota are located in the eastern part. This separation appears to correspond to the Tennessee River Valley and the geological barrier that it represents for the genus, and other co-distributed taxa (e.g. Nannaria wilsoni spe- cies group; Hennen et al. 2022). However, in the southern part of the geographical distribution of Cherokia, especially in the state of Alabama, both shapes of the para- nota overlap and no clear geographical separation was observed (Fig. 11). When this character was traced on the phylogeny of the genus, most individuals in one clade exhibited straight paranota (Fig. 4, blue), while the other clade (and two individuals from Monte Sano State Park, Alabama) possessed sinuate paranota (Fig. 4, red). One individual in the phylogeny and geographical distribution appears as an outlier for the general trend of this character (Fig. 4, GA-FLO-MPE03260*). Although a qualitative character and correlated with metazonite width (p = 0.0001), in some cases it is dif- ficult to distinguish straight versus sinuate, and the variation appears to be clinal. In contrast with the scapulora and color characteristics, this character is largely concord- ant with the phylogeny, but in itself as a single character, insufficient for species or subspecies delimitation. The coloration patterns were plotted on a map to assess concordance with the geographical distribution. Fig. 12 shows the distribution of the patterns (bimaculate, trimaculate, or striped), and the colors (red, orange or yellow). Some localities have all three types of patterns and/or colors—in contrast with Hoffman’s (1960) supposi- tion that each coloration is geographically isolated. Nearly all possible combinations of colors and patterns were observed, but the trimaculate yellow color morph was the most common (both in frequency of individuals and geographical area). The bimacu- late pattern was only observed with an orange hue (the bimaculate orange color morph, Fig. 10A—C). Fig. 12 shows that neither the pattern (bimaculate, trimaculate, striped) nor the colors (red, orange, yellow), have any clear geographical association. [Note that the number of geographical data points that were used for these maps (Fig. 11) were greater (V = 124) than the one used for the phylogenetic analysis (V = 106). Because the number of images available for the specimens actually used in the phylogeny was relatively small (V = 26) and limited the scope of inference, iNaturalist reports for Cherokia were also included in this section.] Perception of color can be affected by the observer, lighting conditions, veiling conditions, and distance, thereby adding error to 158 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) he il 5 @ Sinuate /\_| Straight —— = 0 47 94 141 km Figure I 1. Geographical distribution of Cherokia Chamberlin, 1949, showing the two types of paranota shape. Map includes specimens used for the morphological analysis and deposited at VTEC (N = 105). the evaluation of this character (Endler 1990). In the future, a less error-prone and less human-centric technique should be implemented to obtain more accurate coloration data such as using a spectrometer and incorporating the visual systems of the predators of Cherokia (likely avian) to evaluate the coloration according to the perceivers’ eyes. The use of citizen science as a tool for obtaining and analyzing data has been suc- cessfully demonstrated by various research groups. The Cornell Lab of Ornithology, for example, has developed multiple projects involving amateur ornithologists and the general public for around two decades. Data obtained from those initiatives have been published in several peer-reviewed research papers in various journals (Bonney et al. 2009). The small-scale citizen science project that was made as part of this research demonstrated that it is an effective method to obtain samples from remote and inacces- sible localities, or in special situations such as the SARS-CoV-2 pandemic. Although the first response to the initiative was highly positive, follow-up contact with the interested participants was more difficult and less successful. The number of people who shipped samples back to us (V = 12) corresponds to around the 30% of the kits shipped to selected participants (V = 41). Improved communication with the partici- pants, and a more structured timeline will be needed to increase the overall success of this initiative in future projects. Nonetheless the citizen science project offered an impactful opportunity to share the research with a broader community. Phylogenetic review of the millipede genus Cherokia 159 me 7 ae \_ West Virginia @ Striped bg pit tape a A Kentucky Pal Virginia A Timaculate =a = ex - 4 = 40-6 , - ( \ Be Tay Wy @ Ady \ a . as *% > die A e \ ‘A A ‘ ° \ A A , \ | ee | \ a C) South Carolina a *a\ f \ A Alabama A 5 a A \A Georgia A ( | % | } | / | ( | \ | a a raf ) Florida a cn. ee is West Virginian; wn ® Red ~B Kentucky ss 7 Virginia A Orange Md BB vetiow } A ia? “a ( Tenne Zw ecg ia ial A eu AA, " A a a aH Ae \ | A A \ a "BSS South Carolina A x A % a, \" sa @ ke | ® de S A\ te | \ @ * | \ | Alabama A \ * | Ry eorgia | ® \ e A | rod ( | \ J | \ | é Th ‘Ns \ —~ | S Florida =~ Si ihe fi. S Db cts tf Figure 12. Geographical distribution of Cherokia Chamberlin, 1949 vs. coloration patterns A patterns B colors. Mapped using iNaturalist pictures reported for Cherokia (IN = 124). Conclusions Morphological characters showed clinal variation and a direct relationship with geographical distribution and elevation, but not with the phylogeny. Coloration was highly variable and did not accord with neither geography nor phylogeny. The phylogeny recovered Cherokia as a monophyletic taxon, and the ABGD species 160 L. Fernanda Vasquez-Valverde & Paul E. Marek / ZooKeys 1106: 141-163 (2022) delimitation test showed no barcode gap supporting the existence of multiple species. The molecular and morphological evidence showed that Cherokia is a monospecific genus with the sole species Cherokia georgiana being geographically widespread and highly variable in its morphology. Acknowledgements This research was supported by a National Science Foundation grant to P. Marek (Di- vision of Environmental Biology, Systematics and Biodiversity Sciences # 1916368). Derek Hennen and Jackson Means helped confirm identifications of Cherokia observa- tions on iNaturalist. 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Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1106.81386.suppl1 Phylogenetic review of the millipede genus Cherokia 163 Supplementary material 2 Cherokia georgiana specimens examined Authors: Luisa Fernanda Vasquez-Valverde, Paul E. Marek Data type: List of taxa Explanation note: List of Cherokia georgiana specimens examined from literature and natural collections. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1106.81386.suppl2 Supplementary material 3 Individual gen trees Authors: Luisa Fernanda Vasquez-Valverde, Paul E. Marek Data type: Phylogenetic Explanation note: Individual gene phylogenies of Cherokia georgiana. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys. 1106.81386.suppl3