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Data from: HyRAD-X, a versatile method combining exome capture and RAD sequencing to extract genomic information from ancient DNA
Over the last decade, protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been widely developed. Their use is however limited to DNA of relatively high molecular weight. During the last year, several methods exploiting hybridization capture using probes based on RAD-sequencing loci have circumvented this limitation and opened avenues to the study of samples characterized by degraded DNA, such as historical specimens. Here, we present a major update to those methods, namely Hybridization capture from RAD-derived probes obtained from a reduced eXome template (hyRAD-X), a technique applying RAD-sequencing to messenger RNA from one or few fresh specimens to elaborate bench-top produced probes, i.e., a reduced representation of the exome, further used to capture homologous DNA from a samples set. In contrast to previous hybridization-capture methods, the reference catalog on which reads are aligned does not rely on de novo assembly of anonymous RAD-sequencing loci, but on an assembled transcriptome obtained from RNAseq data, thus increasing the accuracy of loci definition and Single-Nucleotide-Polmorphisms (SNP) call, and targeting, specifically, expressed genes. Finally, the capture step of hyRAD-X relies on RNA probes, increasing stringency of hybridization, making it well suited for low-content DNA samples. As a proof of concept, we applied hyRAD-X to subfossil needles from the coniferous tree Abies alba, collected in lake sediments (Origlio, Switzerland) and dating back from 7200-5800 years before present (BP). More specifically we investigated genetic variation before, during, and after an anthropogenic perturbation that caused an abrupt decrease in Abies alba population size, 6500-6200 years BP. HyRAD-X produced a matrix encompassing 524 exome-derived SNPs. Despite a lower observed heterozygosity was observed during the 6.500-6.200 years BP time slice, genetic composition was nearly identical before and after the perturbation, indicating that re-expansion of the population after the decline was driven by autochthonous specimens. To the best of our knowledge, this is the first time a population genomic study incorporating ancient DNA samples of tree subfossils is conducted at a moderate cost using reproducible exome-reduced complexity.
FIGURE 6 in Mitochondrial DNA sequence analysis of the spectacled salamander, Salamandrina terdigitata (Urodela: Salamandridae), supports the existence of two distinct species
FIGURE 6. Palaeogeographic scenario (Scenario B). (1) The Tyrrhenian basin in the Tortonian time (modified from OrszagSperber et al., 1993) with a land bridge connecting Sardinia with the peninsula. (2) Separation of the CalabroPeloritan massif (CPm) from Sardinia (modified from Duermeijer et al., 1998). Arrows hypothesize dispersal routes. See the text for comments.
FIGURE 5 in Mitochondrial DNA sequence analysis of the spectacled salamander, Salamandrina terdigitata (Urodela: Salamandridae), supports the existence of two distinct species
FIGURE 5. Palaeogeographic scenario (Scenario A). The Italian Peninsula in the Pliocene (modified from Pinna, 1989). Black triangles: sampling sites of Salamandrina populations included in the PER clade. Black stars: sampling sites of Salamandrina populations included in the TER clade. See text for details and confront to the Fig. 1.
FIGURE 4 in Mitochondrial DNA sequence analysis of the spectacled salamander, Salamandrina terdigitata (Urodela: Salamandridae), supports the existence of two distinct species
FIGURE 4. Haplotype network of Salamandrina terdigitata, reconstructed using parsimony probability as implemented in TCS. Each circle in the network corresponds to one observed haplotype. Size of circles is proportional to the number of individuals (in parentheses) carrying a given haplotype. The three letters codes of the populations are indicated; numbered codes in italic mean different individuals when more than one haplotype was found in a population.
FIGURE 3 in Mitochondrial DNA sequence analysis of the spectacled salamander, Salamandrina terdigitata (Urodela: Salamandridae), supports the existence of two distinct species
FIGURE 3. Maximum likelihood tree for Salamandrina terdigitata samples plus outgroup, based on combined 12S, 16S and cytb. HKY85+ model (ln = 2864.386; shape parameter = 0.1203) was assumed. Numbers in boxes (from top to bottom) are bootstrap support values for ML (100 replicates), MP (1000 replicates) and NJ (1000 replicates) and posterior probability percentages in the Bayesian analysis (2 million generators). Specimens are identified by code of sampling locality (as in Tab.1) and by a progressive number.
FIGURE 1. A in Mitochondrial DNA sequence analysis of the spectacled salamander, Salamandrina terdigitata (Urodela: Salamandridae), supports the existence of two distinct species
FIGURE 1. A female specimen of Salamandrina terdigitata from Lepini Mountains (Latium, Central Italy).
FIGURE 20 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 20. Phylogram of a maximum likelihood analysis of the DNA sequence data (Appendix 4) using the general time reversible model. The analysis includes the subaptera – group taxa and one outgroup.
FIGURE 19 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 19. Strict consensus tree (length 374 steps, CI 0.94, RI 0.90) of a maximum parsimony analysis of the DNA sequence data (Appendix 4). Maximum parsimony bootstrap values> 50 % are shown above branches (2000 replications). The analysis includes the subaptera – group taxa and one outgroup.
FIGURE 17 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 17. Distribution of the bisexual species of the subaptera-group: Phyllodromica iberica morph #1, #2 and #3, and P. quadracantha; these species only occur on the Iberian Peninsula. In cases where two different symbols partly overlap, two P. iberica morphs are found at the same locality; in one case all three morphs occur together (Fig. 15, Sp 510 ca. 1° W, 40° N). P. iberica morph #3 symbols labeled with a "+" (Fig. 15, Sp 335) or an " " (Fig. 15, Sp 186, 270, 469 and 512) indicate the presence of morphological variations. At some localities no males have been found ("sad face" symbol) but females with spermathecae containing sperms and/or with oothecae containing male and female offspring. Both facts indicate the presence of a bisexual species of unknown specifity.
FIGURE 16 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 16. Distribution of bisexual species of the subaptera-group and the parthenogenetic species P. subaptera on the Iberian peninsula.
FIGURE 14 Phyllodromica quadracantha, male. A in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 14 Phyllodromica quadracantha, male. A membrane glands of the right lateral region of the anterior border of tergite 6. B right paraproct with bulge on the medio-anterior process (compare Fig. 5 D). C distal end of tibia from the right mid leg (bearing 4 distal tibia spines; compare Fig. 5 E) in posterior view. D helmet sclerite, the nomenclature "fr" and "re" do not indicate the orientation of the sclerite within the animal. E–G tergites 6 (E), 7 (F) and 8 (G) of specimen with slightly different tergal structures on tergite 7. Due to unnatural squeezing of the tergite 8 (G) during the mounting procedure the distance between the anterior processes appears broader than under natural conditions. Abbreviations: fr "frontal" part of helmet sclerite, bf bristle field, bu bulge, hs helmet sclerite, mg membrane glands, mp medio-anterior process of right paraproct, re "rear" of helmet sclerite, r ridge, rp right paraproct, sp spinelike process, tar tarsus, tib tibia, tr transversal trough. Same scale for (A–D) and (F–G). Identification: (A, E–G) Sp 504a/M1, (B, C) Sp 203d/ M1(holotype). (D) Sp 203b/M7.
FIGURE 10 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 10. Phyllodromica iberica morph #3. A–F male, SEM pictures of different tergal glandular structures in dorsal view. (A, B), (E, F) Two different forms of glandular structures of tergites 7 and 8 of two specimens; white arrow heads in A point to the bulge-like cuticule limitations of the antero-lateral borders of the bristle fields which is absent in the other form (white arrow heads in E); on tergite 8 bristles are also present in the central region of the tergite (white arrow heads in B, F); (C, D) Medio-anterior part of tergite 8 of two further specimens showing variously developed conelike processes. The unnatural shape of the tergite in B (compared with F) is due to an mounting artefact. G, H female. (G) Thoracal nota; (H) abdominal tergite 5. Abbreviations: a artificial structures (originated during SEM preparation procedure), bf bristle field, cp conelike process, r ridge between bristle fields, tt transversal trough. Same scale for (A, E), (B, F) and (C, D). Identification: (A, B) Sp 388/M1, (E, F) Sp 512/M1, (C) Sp 186/M7, (D) Sp 361/M2, (G, H) Sp 380/W1.
FIGURE 11 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 11. Phyllodromica iberica morph #3, male. A Tergite 8; B-E Tergite 6; in C, enlargement of the left half of the tergite depicted in B with narrow transversal torus-like structure (between white arrows) bearing bristles (white spots) in enhanced density; in D, lack of torus-like structure, only bristles (white spots) in enhanced density. D, E Tergite 6, two examples of different tergite colouration. F–H Abdominal tergites 6–8 of one specimen. (F) Tergite 6; (G) tergite 7, bristle fields antero-lateral without bulge-like cuticule limitations (white arrows); (H) tergite 8, conelike process nearly completely missing. I–L Abdominal tergites 6–8 of one specimen. (I) Tergite 6; (K) tergite 7, white arrows point to the shallow pouches appearing as crescent–shaped black shadows; (L) tergite 8, well developed conelike process. Same scale for (A, G, H, K, L) and (B, D, E, F, I). Identification: (A) Sp 395/M1, (B, C) Sp 148b/M1, (D) Sp 460/M1, (E) Sp 387a/M1, (F, G, H) Sp 270a/M1, (I, K, L) Sp 335/5.
FIGURE 9 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 9. Phyllodromica iberica morph #3, male. A Thoracal nota. B–F Abdominal tergites 5–9. (B) Tergite 5; (C) tergite 6; (D) tergite 7 and (E) tergite 8 with glandular structures in the anterior part of the tergites; (F) tergite 9. G Terminalia with tergite 10 (sa), cerci and paraprocts. H Hook of left phallomere with the posterior end on the top. I Subgenital plate with remaining genital sclerites (without hook). Abbreviations: a apodemes of subgenital plate, ap anterior process, bf bristle field, c cercus, cl claw of hook, cs cleft sclerite, ea endophallic apodeme, hs helmet sclerite, ml median lobe, rp right paraproct, R3 sclerite of the right phallomere, s stylus, sa supraanal plate, sh shaft, st stalk, sub subgenital plate, tt transversal trough, v velum. Same scale for (B, C, G) and (D, E, H). Identification: Sp 380/M2.
FIGURE 8. A–E in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 8. A–E Phyllodromica iberica morph #2, male. (A–D) Different colour patterns of tergite 6; (E) helmet sclerite. F, G Phyllodromica iberica morph #2, female. (F) Thoracal nota; (G) abdominal tergite 5. H Right front leg of Phyllodromica iberica morph #1, male in frontal view. I Right front leg of Phyllodromica quadracantha, male in frontal view. Abbreviations: fr "frontal" part of helmet sclerite, cox coxa, fem femur, hs helmet sclerite re "rear" of helmet sclerite, tib tibia, tm tegmen. Same scale for (A–D), (F, G) and (H, I). Identification: (A) Sp 266/2, (B) Sp 365/3, (C) Sp 365a/M14, (D) Sp 365a/M8, (E) Sp 267c/M1, (F, G) Sp 267c/W2, (H) Sp 510/M11, (I) Sp 203d/M1 (holotype)..
FIGURE 7 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 7. Phyllodromica iberica morph #2, male, SEM pictures of tergite 6 and the tergal glandular structures. A–C Tergite 6. (A) Latero-frontal view from slightly above, white arrows point to two shallow longitudinal depressions posteriorly of the torus (to); (B) dorsal view of the whole tergite; (C) transversal torus with bristles (between arrows) of the right half of the tergite. As a result of the preparation procedure for SEM the posterior margin of the tergite and its emargination in the middle (in B) appear narrower than under natural conditions. D, E Tergite 7. (D) Lateral view of tergite from slightly above; (E) glandular region in dorsal view. F–H Tergite 8. (F) Dorsal view of the whole tergite with median emargination in the distribution of the bristles (white arrow heads); (G) latero-frontal view from slightly above; (H) lateral view of the anterior part of the tergite. Abbreviations: bf bristle field, cm central mound, cp conelike process, m mound, se sinusoidal edge, sp shallow pit, to transversal torus, tr transversal ridge, tt transversal trough. Same scale for (A, B) and (D, F, G). Identification: (A–C) Sp 267b/M6, (D–H) Sp 267b/M4.
FIGURE 12 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 12. Phyllodromica quadracantha, male (holotype). A Thoracal nota. B–F Abdominal tergites 5–9. (B) Tergite 5; (C) tergite 6; (D) tergite 7 and (E) tergite 8 with glandular structures in the anterior part of the tergites; (F) tergite 9. G Terminalia with tergite 10 (sa), cerci and paraprocts. H Hook of left phallomere with the posterior end on the top. I Subgenital plate with remaining genital sclerites (without hook). Abbreviations: ap anterior process, bf bristle field, c cercus, ml median lobe, rp right paraproct, sa supraanal plate, tr transversal ridge, tt transversal trough. Same scale for (B, C, G) and (D, E, H). Identification: Sp 203d/M1 (holotype).
FIGURE 13 Phyllodromica quadracantha. A–C in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 13 Phyllodromica quadracantha. A–C male, SEM pictures of glandular structures of tergites 7 and 8 in dorsal view. (A) Tergite 7; (B) tergite 8, bristles are present also in the median tergite region (white arrow heads); (C) anteriormedian region of tergite 8, no conelike process present. D–H female. (D) Thoracal nota; (E) Tergite 5; (F) Subgenital plate; (G) dorsal complex of genitalia in ventral view, posterior end on top, without additional sclerite between pl and is (compare Fig. 2 D, E); (H) ventral komplex of genitalia: laterosternal shelf with intersternal folds between the arms. Abbreviations: a apodemal process, bd dorsal sclerite of basivalvula, bv ventral sclerite of basivalvula, c cercus, i intersternal fold, is intercalary sclerite, l laterosternal shelf, ls laterosternite IX, pl posterior lobe of valvifer II, pp paraproct, pt paratergites, tr transversal ridge, T9 tergite 9, T10 tergite 10, v valves. Same scale for (D, E). Identification: (A–D) Sp 499/M1, (E–I) Sp 203d/W1.
FIGURE 6 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 6. Phyllodromica iberica morph #2, male. A Thoracal nota. B–F Abdominal tergites 5–9. (B) Tergite 5; (C) tergite 6 with a narrow transversal torus (white arrow heads) bearing bristles (white spots) in extraordinary density; (D) tergite 7, white arrow heads point to the shallow pouches appearing as crescent–shaped black shadows; (E) tergite 8; (F) tergite 9 on glass rod. G Terminalia with tergite 10 (sa), cerci and paraprocts. H Hook of left phallomere with the posterior end on the top. I Subgenital plate with remaining genital sclerites (without hook). Abbreviations: ap anterior process, bf bristle field cm central mound, cp conelike process, m mound, ml median lobe, mp medio-anterior process of right paraproct, r ridge between bristle fields, rp right paraproct, sa supraanal plate, se sinusoidal edge, sp shallow pit, tt transversal trough. Same scale for (B, C, G) and (D, E, H). Identification: Sp 267c/M2.
FIGURE 5 in Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding)
FIGURE 5. Phyllodromica iberica morph #1, A–E, male. (A) Tergite 7 and (B) tergite 8 of a male specimen differing from the holotype in its colouration; (C) membrane glands of the left lateral region on the anterior border of tergite 6; (D) right paraproct; (E) distal end of tibia of the right mid leg (bearing 5 distal spines) in posterior view. F–I, female. (F, H) Different colour pattern of pronotum; (G, I) tergite 5. Abbreviations: mg membrane glands, mp medio-anterior process of right paraproct, rp right paraproct, sp spinelike process of right paraproct, tar tarsus, tib tibia. Same scale for (C, D) and (F–I). Identification: (A, B) Sp 330/3, (C, D) Sp 292b/M3 (holotype), (E) Sp 510/M1, (F, G) Sp 292b/W1, (H, I) Sp 292b/W2.
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