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Figures 22–31.Hermeuptychia intricata. 22–23 holotype, others are paratypes, data in text and Table 1. Sexes and DNA voucher codes are: 24 ♀ NVG-1554 25 ♀ NVG-1565 26–27 ♀ 13385G09 28–29 ♂ NVG-1631 30–31 ♀ NVG-1629. Dorsal/ventral surfaces are in even/odd-numbered figures, except 24, which is ventral. Labels are shown for the holotype and are reduced 2.5-fold compared to specimens as indicated by a smaller scale bar. “F” specifies mirror image (left-right inverted).
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Figure 68.DNA-barcoded Hermeuptychia specimens from USA: Texas: Fort Bend Co., Brazos Bend State Park. Hermeuptychia intricata is above the line and Hermeuptychia sosybius is below the line, photographed prior to removal of body parts for DNA extraction. DNA voucher codes (see Table 1 for data) are shown below each specimen. Hypothetical field marks are indicated on the first specimen of each species. NVG-1537–NVG-1553 are from Horseshoe Lake trail, 29°22'54.96", −95°36'41.06", 15 m; and NVG-1554–NVG-1567 are from near Hale Lake, 29°22'48.27", −95°35'05.02", 16 m, all collected on 17-Aug-2013. Both species are present in each locality. Images are scaled approximately. “F” specifies mirror image (left-right inverted).
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Figures 32–47.Hermeuptychia intricata paratypes and Hermeuptychia sosybius specimens. 32–35, 40–43 Hermeuptychia intricata 36–39, 44–47 Hermeuptychia sosybius; data in text and Table 1. Sexes and DNA voucher codes are: 32 ♂ 13385G11 33 ♀ 13385G08 34 ♂ 13385G07 35 ♂ 13386A02 36 ♂ 13385G10 37 ♀ 13385G12 38 ♀ 13386A04 39 ♀ 13386A06 40 ♂ 13385H02 41 ♂ 13385H01 42 ♂ 13386A05 43 ♂ 13386A03 44 ♂ 15609E04 45 ♀ 13385H07 46 ♂ 13385H08 47 ♂ 13386A01. All specimens are in USNM collection. Ventral wing surfaces are shown. “F” specifies mirror image (left-right inverted).
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Figure 60.Male genitalia of Hermeuptychia from USA: Texas. a, d, g, j Hermeuptychia sosybius, Fort Bend Co., Brazos Bend State Park, Horseshoe Lake trail, 29°22'54.96", −95°36'41.06", 15 m, 17-Aug-2013, leg. N. V. Grishin, DNA voucher NVG-1542, genitalia NVG130927-03 (forewing length 15 mm) b, e, h, k Hermeuptychia hermybius sp. n. paratype, Cameron Co., E of Brownsville, 18-Jan-2003, leg. N. V. Grishin, DNA voucher NVG-1607, genitalia NVG130927-18 (specimen Figs 54–55, forewing length 15.5 mm) c, f, i, l Hermeuptychia intricata sp. n. holotype, Fort Bend Co., Brazos Bend State Park, near Hale Lake, 29°22'48.27", −95°35'05.02", 16 m, 17-Aug-2013, leg. N. V. Grishin, DNA voucher NVG-1560, genitalia NVG130927-14 [USNM] (specimen Figs 22–23, forewing length 16.5 mm). Views: a–b dorsal, perpendicular to the tegumen-uncus-gnathos plane c–d ventral, perpendicular to the plane of saccus and valvae (appears larger than dorsal view due to different projection axis) e–f right dorsolateral g–h right lateral. All images are to scale. Diagnostic characters are indicated on images. Note that Hermeuptychia intricata with larger than Hermeuptychia sosybius wings has smaller genitalia.
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Figure 61.Dorsoposterior view of male abdomens of Hermeuptychia from USA: Texas. a Hermeuptychia intricata, DNA voucher NVG-1548 (mirror image, i.e. left-right inverted) b Hermeuptychia hermybius, DNA voucher NVG-1635 (also shown in Fig. 62j, specimen Fig. 52) c Hermeuptychia sosybius, DNA voucher NVG-1553. Data in Table 1. Scales are brushed off the abdomen tip to expose distal parts of genitalia. The easiest to observe character (the shape of the distal end of uncus) is indicated.
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Figure 62.Variation in male genitalia of Hermeuptychia hermybius and Hermeuptychia sosybius. a–m Hermeuptychia hermybius paratypes, DNA (or genitalia, where DNA sequence is not available, and full data for these given) voucher codes: a. NVG-1609 b NVG-1610 c Texas: Cameron Co., Brownsville {10-13}-Mar-1979, T. Friedlander, NVG140104-01 d NVG-1698 e NVG-1699 (specimen Fig. 56) f NVG-1714 g NVG-1726 h NVG-1727 i NVG-1747 j NVG-1635 (also shown in Fig. 61b, specimen Fig. 52) k 13385H10 (specimen Fig. 53) l–m Mexico: Tamaulipas, leg. R. O. & C. A. Kendall: l Quintero cave, 7-Jan-1974, NVG130104-24 (specimen Fig. 59) m Ciudad Mante, Los Arcos Ct., 19-Dec-1973, NVG130104-23 (specimen Fig. 58) n Hermeuptychia intricata paratype, NVG-1631 (specimen Figs 28–29), diagnostic characters are indicated on the image o–z2 Hermeuptychia sosybius: o 13386A01 (specimen Fig. 47) p 13386A07, neotype (specimen Figs 9–11) q 13385H04 r 13385H08 (specimen Fig. 46) s 13385H03 t NVG-1550 u NVG-1546 v NVG-1559 w NVG-1566 x NVG-1630 y NVG-1633 z Texas: Marion Co., W of Caddo Lake, 5-Apr-1997, leg. N. V. Grishin, NVG-1634 z1 NVG-1632 z2 Texas: Wise Co., LBJ National Grassland, ex ovum, eclosed 3-Aug-1998, leg. N. V. Grishin, NVG-1604. c, l, m are in TAMU and o–s are in USNM collections. Data for most specimens are in Table 1, text, or specified above. Complete genitalia are shown in lateral view, and dorsal view of uncus is shown above and to the right of each specimen. Aedeagus is shown below in k DNA (or genitalia, where DNA sequence is not available) voucher codes and general localities are indicated below each genitalia image. “F” specifies mirror image (left-right inverted).
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Figure 64.Antrum in female genitalia of Hermeuptychia from USA: Texas. a–h Hermeuptychia sosybius, Fort Bend Co., Brazos Bend State Park, 17-Aug-2013, leg. N. V. Grishin: a–f is from Horseshoe Lake trail, 29°22'54.96", −95°36'41.06", 15 m and g–h is from near Hale Lake, 29°22'48.27" −95°35'05.02", 16 m; DNA voucher|genitalia dissection codes are: a–b NVG-1537|NVG130927-01 c–d NVG-1540|NVG130927-02 (specimen Fig. 12) e–f NVG-1552|NVG130927-06 g–h NVG-1562|NVG130927-10 i–p Hermeuptychia intricata sp. n. paratypes, Fort Bend Co., Brazos Bend State Park, near Hale Lake, 29°22'48.27", −95°35'05.02", 16 m, 17-Aug-2013, leg. N. V. Grishin; DNA voucher|genitalia dissection codes: i–j NVG-1554|NVG130927-07 (specimen Fig. 24) k–l NVG-1558|NVG130927-08 m–n NVG-1563|NVG130927-11 o–p NVG-1565|NVG130927-12 (specimen Fig. 25) q–z Hermeuptychia hermybius sp. n. paratypes q–r Cameron Co., E of Brownsville, ex ovum ex ♀ captured on 18-Jan-2003, eclosed on 17-Mar-2003, leg. N. V. Grishin, NVG-1603|NVG130927-17 s–t ibid., eclosed on 14-Mar-2003, NVG-1611|NVG131017-03 u–v ibid., eclosed on 16-Mar-2003, NVG-1612|NVG131017-04 w–x TX: Hidalgo Co., 1.5 air mi SE of Relampago, Rio Rico Rd., 26.07, -97.891, 21 m, 19-Oct-2013, leg. W. R. Dempwolf, NVG-1695|NVG131229-03 y–z Starr Co., 0.5 mi S of Fronton, 26.399, -99.085, 50 m 10-Oct-2013, leg. W. R. Dempwolf, NVG-1737|NVG131229-11 (specimen Fig. 57). Additional data for specimens and their DNA barcodes are in Table 1. In all images, posterior end is pointing up (i.e. ostium bursae is closer to the top of each image); a, c, e, g, i, k, m, o, q, s, u, w, y are in lateral view, others are in right ventrolateral view. All images are to scale. Diagnostic characters to tell between Hermeuptychia sosybius and Hermeuptychia intricata are indicated on images, each character was invariantly observed in all inspected samples of a species, but is indicated (for clarity) on a single image only. We failed to find characters distinguishing female genitalia of Hermeuptychia hermybius from Hermeuptychia sosybius and simply illustrate genitalic variation.
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Figure 65.DNA ID tags of specimens that are over 100 years old. ID tag #2 is shown as an example. The tag region sequence alignment of the three species: Hermeuptychia sosybius, Hermeuptychia hermybius, and Hermeuptychia intricata is shown in the middle and positions at which sequences differ are highlighted in cyan and boxed. Each of the three species differs from the other two by at least 2 nucleotides, and Hermeuptychia sosybius is different from Hermeuptychia intricata by 4 nucleotides. Forward and reverse primer regions are shaded. DNA of the tag was amplified and sequenced in both forward and reverse directions from two over-100-years-old specimens from the same locality (SC: Clarendon Co.). Forward and reverse sequences traces for the first specimen are shown above the reference sequences and the two traces for the second specimen are shown below. It is clear from the traces that the specimen above (13385G10, Fig. 36) is Hermeuptychia sosybius, (A, T, T, & C at these 4 positions, no contamination seen) and the one below (13385G08, Fig. 33) is Hermeuptychia intricata (G, C, C, & T at these 4 positions and equally unambiguous traces). Nucleotides that identify each specimen are indicated in large letters on yellow background and arrows point to the trace peaks revealing these nucleotides. This strategy was applied to identify 12 very old specimens of three species in a random order and yielded unambiguous identifications for 11 of them. One sample appeared to be contaminated, and the traces showed the presence of several nucleotides in many positions. All 11 DNA-based identifications agreed with genitalic identifications.
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Figure 66.DNA-derived data. a Analysis of named Hermeuptychia species b relationships between Hermeuptychia specimens from USA in a form of BioNJ (Dereeper et al. 2008) distance tree. a DNA barcode distance matrix is shown on the right and a BioNJ distance tree corresponding to it is on the left. The tree is rooted with Megisto cymela (Cramer, 1777) sequence. A more comprehensive tree that includes several specimens of each species (except those described herein) and their detailed analyses are given in Seraphim et al. (2014) and is not repeated here. Only a single representative sequence for each species is used in a for clarity. The scale bar corresponding to about 1% difference in sequences is placed below the tree. Bootstrap support values are shown by each node in the tree; values below 0.5 indicate possibly incorrect groupings. GenBank accessions (http://genbank.gov/) for sequences are given after species names. NT, PT and HT refer to neotype, paratype and holotype, respectively. Data for specimens are in Table 1. Specimens 4–11 were not examined and their identification follows that of the authors who performed sequencing studies and analyses (Peña et al. 2010, Silva-Brandão et al. 2011, Seraphim et al. 2014). Percent difference and the number of different nucleotides are shown below and above the diagonal in the matrix, respectively, and the length of each sequence segment (bp) used in the analysis is on the diagonal. Most instructive values discussed in the text are shown in bold font b GenBank accession numbers (those that start with letters G or K) for sequences retrieved from GenBank or DNA voucher numbers (those that start from a number or letter N) for sequences obtained in this study and locality data for specimens are given for each sequence. Further details about the specimens are provided in Table 1. Hermeuptychia sosybius specimens with sequences obtained from GenBank were not examined and their identification follows that of the authors who performed sequencing studies (Murray and Prowell 2004, Hebert et al. 2010 & Seraphim et al. 2014). All Hermeuptychia hermybius and Hermeuptychia intricata specimens are paratypes, except the holotype marked with “HT”. Scale bar shown below indicates about 0.5% difference.
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Figure 67.USA localities of Hermeuptychia specimens with available DNA barcode information. Color of circles corresponds to species: Hermeuptychia sosybius – black; Hermeuptychia hermybius – blue, Hermeuptychia intricata – red, split red/black circles mark localities where both Hermeuptychia sosybius and Hermeuptychia intricata were recorded. Type localities are indicated with a corresponding name followed by “TL”. Hermeuptychia hermes kappeli was treated as a junior subjective synonym of Hermeuptychia sosybius by Pelham (2008). DNA barcode of Hermeuptychia hermes kappeli holotype is 100% identical with the barcode of Hermeuptychia sosybius neotype. DNA barcode amplification failed for Hermeuptychia intricata specimen from LA: Jonesboro and for Hermeuptychia sosybius specimen from TX: Brazoria Co., and their identification is based on genitalia only. Specimens from all localities except those from TN and NC (data from GenBank, specimens not inspected) and from FL: St. Petersburg (specimen lacked abdomen) were dissected, and genitalia-based identification agreed with DNA barcode-based identification in all cases (see Fig. 66).