Ecology

The present study compared DNA sequences in a whole genome between the long strain and standard genome samples as references or the short strain and standard ones in T. castaneum. The results of resequencing analysis showed variations of DNA sequence from the reference sequence in both long and short strains, and the variations were detected more frequently in the long strain in a whole genome. Small nucleotide variants (SNV), multi-nucleotide variants (MNV), deletion, insertion, and replacement were detected in a whole genome in long and short strains. The same DNA sequence variants sharing between long and short strains were removed for the analyses. The numbers of small variants in total were larger in long strains than short strains (Fig. 1, Tables S1 and S2). The most frequent type of small variants was SNV, and the proportions of SNV were 82.7% (93,233/112,783) in long strains and 82.8% (13,817/16,697) in short strains, respectively (Fig. 1A). The SNVs compared with the reference nucleotide occurred frequently between adenine and guanine or cytosine and thymine in both long and short strains (Fig. 1B), and the frequencies were up to three times as large as other base combinations, indicating more frequent transition and fewer transversion variants. Deletion and insertion ranged from one to nine bases in both long and short strains, with one base was frequently deleted or inserted (Fig. 1C). Homozygosity presented more frequently than heterozygosity in all linkage groups, but the rate of homozygosity to heterozygosity depended on the linkage groups (Fig. 1D). Homozygosity of variants was more frequent in linkage groups 3 (LG3), 5 (LG5) and 7 (LG7) than other linkage groups in both strains. The ratios of homozygosity to heterozygosity were the largest in LGX and LG2 in long and short strains, respectively.Figure 1Analytical results of small variants of DNA sequence in a whole genome level in long and short strains. Proportion of small variants as SNV, MNV, deletion, insertion, and replacement in long and short strains (A). The numbers of small variants are indicated as the diameter of a pie graph. Frequencies of the SNVs in both long and short strains were compared with the reference nucleotide (B). Insertion and deletion ranged from one to nine bases in both long and short strains (C). Frequency of homozygosity or heterozygosity and its ratio in all linkage groups in long and short strains (D).Full size imageThe variants distributed in cording and non-cording regions. Figure 2A shows the results of narrowing down the variants in genic region from the variants in a whole genome in the long and short strains, and then aggregating the variants information in the exon, intron, URT and other regions. In all genic region, numbers of variants were larger in long strain than short strain. Then, genes containing these variants were counted in each strain (Fig. 2B). In exon region, genes with nonsynonymous variants were more numerous in the long strain (3243) than the short strain (844), and 464 common genes containing different DNA sequence variants between the strains were detected (Fig. 2B). In the genes with synonymous variants or the genes with variants in intron or UTR, the numbers of genes in long strain were constantly larger than those in short strain (Fig. 2B). The functions of long-unique, short-unique and common genes with variants were sorted into four categories by enrichment analyses as gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) ongoloty (KO) terms (Fig. 2C, Table S3). In the biological process, cellular component, and molecular function, and KEGG pathway, characteristics of nonsynonymous variants in long-unique, short-unique and common genes did not basically overlap among them, indicating specific selection of gene characteristics for each strain. Characteristics of synonymous variants were also sorted, but the synonymous variants may not influence the amino acid sequence of the gene and structure of the protein translated, rather these characteristics may be necessary to maintain the strain and preserved under artificial selection. Variants in intron and UTR may have potential effects on the gene expression, but should be investigated in detail in future study. Analyses of cis-regulatory elements might be important to understand regulation of gene expression, but the information on this region in T. castaneum is not available, therefore, the variants in cis-regulatory elements could not be analyzed.Figure 2Analytical results of the position of small variants in a whole genome in long and short strains (A) Numbers of variants in genic region including exon region, intron, UTR and other non-cording regions were indicated. As shown in parentheses, some ncRNAs and tRNAs were contained in exon, intron, and UTR regions. In short strain, there were five regions where two different genes overlap in 5′-UTR and 3′-UTR, respectively. Numbers of genes with variants in exon, intron and UTR regions in long and short strains (B). Numbers of long-unique, short-unique and common genes were shown by Venn diagrams. Common genes contain variants with different DNA sequences between long and short strains. Enrichment analyses of the function of genes with variants sorted into four categories (biological process, cellular component, molecular function, and KEGG pathway) (C). The heatmap is generated using the R package “gplots” (version 3.1.1, https://cran.r-project.org/web/packages/gplots/index.html). The list of each ontology shows the ID and term. The KO id is shown by a three- or four-letter organism code, the first-letter of the genus name and the first two- or three-letters of the species name of the scientific name of the organism, with pathway number. For example, Neuroactive ligand-receptor interaction of Tribolium castaneum is shown as “tca04080”.Full size imageTo explore the position of genes with variants associated with duration of death feigning in linkage groups, bulk segregant analysis was carried out (Fig. 3). The red approximate lines of the plot data crossed over the green threshold lines (P  More

Geological backgroundThe Luoping Biota from quarries near Daaozi Village, Luoping County, Yunnan Province, China, includes diverse arthropods, conodonts, foraminifers, molluscs, echinoderms, brachiopods, fishes, marine reptiles, plants, and trace fossils8,10,11,12,13. The fossil beds occur in Member II of the Guanling Formation which in the Daaozi section comprises approximately 16 m of dark-coloured micritic limestone, thin to moderately thickly bedded, indicating a semi-enclosed intraplatform setting10,11. The co-occurring conodont assemblages, primarily consisting of Cratognathodus sp. and Nicoraella kockeli, indicate that the Luoping Biota belongs to the Pelsonian Substage of the middle Anisian, and the U–Pb age, which is 246.6 ± 1.4 Ma, of the volcanic tuff at the bottom of Member I confirms this age10,14.Systematic palaeontologySuperorder Sauropterygia Owen, 186015.Order Eosauropterygia Rieppel, 199416.Family Incertae Sedis.Genus Diandongosaurus Shang, Wu & Li, 2011.Type speciesDiandongosaurus acutidentatus Shang, Wu & Li, 2011.Revised diagnosisSmall-to-medium-sized eosauropterygian with the following unique combination of characters: premaxilla with long, fang-shaped teeth; maxilla with single enlarged fang alongside smaller teeth; parietal foramen about level with anterior margin of supratemporal fenestra; supratemporal smaller than orbit; interorbital bridge broad; frontal excluded from orbit; posterolateral processes of frontal extending over anterior margin of supratemporal fenestra; postorbital excluded from infratemporal fenestra by contact between jugal and squamosal; ectopterygoid present; vertebral column consisting of about 38 presacral, 3 sacral, and more than 30 caudal vertebrae; anterior caudal ribs elongate without tapering distal end; clavicle with distinct anterior processes laterally; entepicondylar foramen absent; acetabular process of pubis strongly offset from the main body.Diandongosaurus cf. acutidentatus.MaterialWIGM SPC V 1105, a nearly complete skeleton exposed ventrally (Fig. 1).Figure 1Full skeleton of WIGM SPC V 1105, viewed from above. Note the missing left foot. Scale bar = 10 cm.Full size imageLocality and horizonDaaozi Village, Luoping County, Yunnan Province, China; Member II of the Guanling Formation, Anisian, Middle Triassic.DescriptionWIGM SPC V 1105 is a large pachypleurosaur with a length of 88.6 cm from the tip of the snout to the end of the caudal vertebral column (Fig. 1). The specimen is exposed in ventral view, with the cranium exposed both ventrally and dorsally. In the holotype, the cranium comprises 7.8% of the total length, neck 22.9%, trunk 32.4%, and tail 36.9% (Table 1).Table 1 Selected measurements (in mm) of WIGM SPC V 1105.Full size tableSkullThe skull of WIGM SPC V 1105 is exposed in both dorsal and ventral views and is dorsoventrally compressed (Fig. 2). The external naris and the supratemporal fenestra are oval-shaped, while the orbit is nearly circular.Figure 2Photograph and interpretative drawing of the skull of WIGM SPC V 1105. (a, b) In dorsal view; (c, d) In ventral view. ang. Angular, at.c atlantal centrum, at.nar atlantal neural arch, ax.c axial centrum, ax.nar axial neural arch, bo basioccipital, d dentary, ec ectopterygoid, eo-op exoccipital-opisthotic, f frontal, hd hyoid, j jugal, m maxilla, n nasal, p parietal, pat proatlas, pl palatine, pm premaxilla, pob postorbital, pof postfrontal, prf prefrontal, pt pterygoid, q quadrate, qrp quadrate ramus of pterygoid, rap retroarticular process, sang surangular, so supraoccipital, sp splenial, sq squamosal, vo vomer. Scale divisions in (a) = 1 mm. Scale bar in (b–d) = 2 cm. The figure is generated using CorelDRAW X7 (https://www.coreldraw.com/en/pages/coreldraw-x7/).Full size imageIn dorsal view (Fig. 2a,b), the premaxillary portion of the rostrum protrudes, defined by snout constriction at the anterior maxilla, different from the reported specimens of D. acutidentatus17,18. The premaxilla forms the anterior and the medial margins of the external naris. The nasal process extends and narrows posteriorly alongside the nasal posteromedially, reaching the anterior margin of the orbit, and contacting the anterior frontal with a cuspidal border line. The premaxilla contacts the maxilla lateral to the external naris.The maxilla is elongate, with a laterally broad anterior portion and tapering posterior process. Its anteromedial margin forms the posterolateral border of the external naris and is overlapped by the posterior premaxilla laterally. The anterior snout constriction is mostly defined by strong medial curvature of the anterolateral maxilla margin. Medially the maxilla contacts the nasal immediately posterior to the external naris, and the prefrontal posterior to that; the nasal contact is likely the longer. Posteriorly, the maxilla borders the anterolateral margin of the orbit. The posterior process of the maxilla contacts the jugal lateral to the orbit. The nasals are broken. They are separated medially by the premaxilla and make a small contribution to the posterior external naris. The external naris is subcircular.The prefrontal is an arch-shaped bone, fused with the lacrimal. Its dorsal portion expands posteriorly, with its ventral portion forming the anterodorsal margin of the orbit. Posteriorly, the prefrontal overlaps the postfrontal obliquely at the midpoint of the dorsal border of the orbit. The postfrontal is a small trapezoid-shaped bone that forms the posterodorsal margin of the orbit, and is more extensive than in Dianopachysaurus dingi19. Posteriorly, it meets the postorbital anterior to the supratemporal fenestra and has a small medial contact with the parietal, separating the postfrontal from the supratemporal fenestra. Both the prefrontal and postfrontal contact the frontal dorsally, preventing it from entering the orbit.The frontals are fused medially into a butterfly shape in dorsal view, expanding obliquely in four directions. Anteriorly the contacts with the nasals are uncertain but were likely to have been broad. The median contact with the premaxilla is narrow and irregular. The frontal meets the prefrontal and the postfrontal laterally along the arc of the dorsal orbital margin, preventing it from entering the orbit, as in Diandongsosaurus acutidentatus17, but unlike both Keichousaurus hui and Dianopachysaurus dingi19,20. The frontal does not enter the supratemporal fenestra either, being narrowly excluded by the parietal and the postorbital as in D. acutidentatus17. In Dianopachysaurus dingi19, contact between the postfrontal and parietal excludes the frontal from the supratemporal fenestra. Posteriorly, the frontal expands slightly, laterally towards the supratemporal fenestrae, and diverges into a narrow fork around the anterior processes of the parietals, separating them from the postfrontal.The parietals are partly fused, showing a suture only anterior to the pineal foramen. The anterior processes insert between the posterior frontal margins with an arch-shaped border. Laterally, the parietal extends a short process to meet the postorbital in a narrow contact at the anterior margin of the supratemporal fenestra, posterior to the postfrontal. This differs from K. hui and Dianopachysaurus dingi19,20, in which the parietal contacts the postfrontal anterolaterally. The bone forms the medial margin of the supratemporal fenestra. The narrow posterolateral processes are inserted by the dorsal processes of the squamosal. The pineal foramen is sub-circular and aligns with the anterior margin of the supratemporal fenestra, more anterior than in K. hui20 and not elongate as in Dianopachysaurus dingi19.The postorbital is roughly triradiate, developing three processes: anteroventral, anteromedial, and posterior. The anteroventral process outlines the posterior border of the orbital, overlapped by the jugal laterally. The narrow anteromedial process extends dorsally, forming the anterior margin of the supratemporal fenestra, and meeting the postfrontal and the parietal anterior to the supratemporal fenestra, unlike in the reported specimens of D. acutidentatus, K. hui, and Dianopachysaurus dingi17,19,20, and more like nothosaurs21,22. It is broadly overlapped by the postfrontal. The posterior process is triangular and extends nearly to the posterolateral margin of the supratemporal fenestra, forming the border of most of its lateral portion. Posteriorly, the tip of the process inserts into the squamosal.The jugal is boomerang-shaped, forming most of the lateral border of the orbit. It contacts the maxilla at the anteroventral margin of the orbital, dorsally overlapping it. Posteriorly, the jugal forms the anterior border of the infratemporal fenestra. Its posterior process is anteroposteriorly broad and extends dorsally, overlapping the postorbital at the posteroventral margin of the orbital. As in D. acutidentatus17, the posterior process of the jugal has a small contact dorsally with the anterior process of the squamosal.The squamosal is a large bone expanded in four directions. The anterior process forms most of the upper temporal bar, extending anterior to the level of the anterior margin of the supratemporal fenestra and partially overlapped medially by the postorbital, except where the squamosal holds the posteriormost point of the postorbital. Anteriormost on the squamosal, there is a small lateral contact with the posterior process of the jugal. The medial process of the squamosal forms almost the whole posterior margin of the supratemporal fenestra, inserting into the posterolateral process of the parietal medially. The posterolateral descending process is robust and expands ventrally, forming a sheet at the posterior margin of the cranium and contacting the lateral portion of the quadrate on its posteromedial face. However, the posterior process, the shortest of these four processes, is not as obvious as in the reported specimens of Dianopachysaurus. acutidentatus or K. hui17,20. The supratemporal fenestra is rounded and smaller than the orbit, with a straighter lateral margin. It is less elongate than in Dianopachysaurus dingi and K. hui19,20.The quadratojugal is not exposed. The supraoccipital is a rhomboid bone inserted ventral to the parietal but is substantially broken; it forms the dorsal margin of the foramen magnum. The exoccipital-opisthotic forms the lateral margin of the foramen magnum, while the basioccipital forms the ventral; these elements are also broken.In ventral view (Fig. 2c,d), the internal choana is roughly circular. The vomer is a long bone with a bifurcating posterior portion along the midline of the palate and forms the medial margin of the internal choana. Anteriorly, the bone meets the palatal portion of the premaxilla and contacts the maxilla anterolaterally. Posteriorly, the posteromedial processes of the two vomers are separated by the anterior process of the pterygoid and the posterior contact with the palatine is small, as in D. acutidentatus18,22 but unlike in K. hui20.The palatine is a strap-like bone. It forms the posterolateral margin of the internal choanae. Anterolaterally, it contacts the maxilla, and meets the vomer on its medial side. Posteromedially, there is a highly irregular, oblique suture line between the palatine and the pterygoid.The pterygoid is one of the largest bones of the skull, forming most of the palate posteriorly. The two pterygoids are fused along the midline leaving a straight groove anteriorly that becomes more irregular posteriorly. Unlike D. acutidentatus, it has neither central opening, nor posterior vacuity18. The tapering anterior process of the pterygoid inserts between the two vomers, whereas it is overlapped in K. hui20, and anterolaterally the pterygoid has a large oblique contact with the palatine. Laterally, the transverse process of the pterygoid expands ventral and posterior to the posterior margin of the ectopterygoid. The pterygoid forms almost the entirety of the subtemporal fenestra margin anteriorly, medially, and posteriorly. The elongate quadrate ramus of the pterygoid extends posterolaterally to the posterior margin of the quadrate, making a long contact with the pterygoid ramus of the quadrate.The ectopterygoid is roughly a small square bone, suturing to the transverse process of the pterygoid. It is not as prominent as in nothosaurs (e.g. Nothosaurus21, Lariosaurus22), but is relatively larger than in the reported specimens of D. acutidentatus18,23, whereas the presence of an ectopterygoid is uncertain in K. hui and Dianopachysaurus dingi19,20. The ectopterygoid contacts the palatine anteriorly, excluding the palatine from the subtemporal fenestra. Posteriorly it makes a small contribution to the subtemporal fenestra margin lateral to the transverse process of the pterygoid. The quadrate is exposed partly, contacting the quadrate ramus of the pterygoid with its pterygoid ramus. Two rod-like hyoids are ossified and well preserved, lying beneath the pterygoid. They are elongate and slightly expanded at each end.MandibleThe mandible is exposed mainly in ventral view and partly in dorsal (Fig. 2). The dentary is a long bone, occupying over one-half of the ramus as a counterpart to the premaxilla, with a laterally broader symphyseal portion than in D. acutidentatus or K. hui18,20,23. The surangular is partly exposed in dorsal view along the dorsal margin of the mandible, extending ventral to the squamosal. The angular is a long strap-shaped bone that meets the dentary anteriorly and the retroarticular process posteriorly. The articular is sutured dorsal to the angular, with a distinct retroarticular process that extends posteriorly with a tapering end.DentitionIn ventral view (Fig. 2c,d), nine premaxillary teeth and seven lower teeth are visible, which are procumbent, fang-like and with apicobasal striations. The 2nd and 3rd right and the 1st, 3rd and 5th left premaxillary teeth are fully grown, elongate and less curved compared to the other teeth. However, the reported specimens of D. acutidentatus and the nothosauroids Lariosaurus and Nothosaurus carry five teeth on each premaxilla17. The space between the 2nd and 3rd right premaxillary teeth suggests that there might be one or two missing teeth. There is one fang-like tooth on each maxilla, surrounded by small tapering teeth, and there are five to six corresponding teeth in the lower jaw. The caniniform teeth also have apicobasal striations like the premaxillary teeth. The row of dentary teeth is restricted to a level anterior to the posterior margin of the orbit.Vertebrae and ribsThere are 38 presacral vertebrae, 3 sacral and 33 caudal (Fig. 1); these counts are roughly the same in coeval Eosauropterygia19,24,25. The atlas and axis are dorsally exposed (Fig. 2a,b). The atlas leans anteriorly, and its neural spine does not meet its counterpart. The proatlas is a pentagonal bone, disarticulated from the atlas. The axis has been rotated laterally, but still articulates with the atlas.There are 19 cervical vertebrae, compared to 20/21 in Dianopachysaurus dingi19. The centra cylinders are rhomboidal in ventral view, increase in length posteriorly and the vertebrae articulate with one another compactly. The parapophyseal articulation on the cervical rib (CR), visible in ventral view, is robust and offset about 90° from the long axis of the rib, defined between the main body and a prominent anterior process. These posterior and anterior extensions are approximately equal in length until about CR14, where the posterior extension starts to lengthen strongly. The anterior process becomes strongly reduced from CR16 onwards.There are approximately 19 thoracolumbar vertebrae, most of which are covered by the gastralia (18 in Dianmeisaurus gracilis25); the count estimated from two gastralial rows corresponding to one vertebra. The intercentral articulation is less compact than in the cervical vertebrae. The transverse processes face posteriorly. The dorsal ribs are single-headed arch-shaped bones with slightly expanded proximal flat ends, but otherwise retain constant diameter along their whole length, ending distally in a flattened stub. Dorsal ribs DR1–6 are exposed ventrally, while the rest are mostly overlain by the gastralia. There are 24 rows of gastralia, suggesting 12 more dorsal vertebrae covered, each gastralium consisting of one medial element and four lateral elements (Fig. 4a).Three sacral vertebrae can be recognized in dorsal view (Fig. 4b), the same as in Dianmeisaurus gracilis, Dianmeisaurus dingi and K. hui19,24,26. The sacral ribs are elongate and cylindrical with thickened distal ends, and closely articulate with the centrum and possibly overlap the rib posterior to each proximally. Distally the sacral rib is expanded posteriorly into a small triangular process that overlaps the next sacral rib posteriorly. Sacral ribs SR2 and SR3 likely articulate with the ilium, while the others are overlain by pubis and ischium (Fig. 3c,d).Figure 3Photographs and interpretative drawings of the pectoral girdle, forelimb, pelvic girdle and hindlimb of WIGM SPC V 1105 in ventral view. (a, b) Pectoral girdle and forelimb. (c, d) Pelvic girdle and hindlimb. as astragalus, cal calcaneum, cl clavicle, co coracoid, cr1 caudal rib 1, cr19 cervical rib 19, cv1 caudal vertebra 1, cv19 cervical vertebra 19, dc2 distal carpal 2, dc3 distal carpal 3, dc4 distal carpal 4, dr2 dorsal rib 2, dv2 dorsal vertebra 2, dr19 dorsal rib 19, dv19 dorsal vertebra 19, f femur, fi fibular, hu humerus, icl interclavicle, il Ilium, in intermedium, is ischium, mc1 metacarpal 1, mc5 metacarpal 5, mt1 metatarsal 1, mt5 metatarsal 5, pu pubis, ra radius, sc scapula, sr1 sacral rib 1, ti tibia, ul ulna, uln ulnare. Scale bar in (a, b, d) = 2 cm. Scale divisions in (a) = 1 mm. The figure is generated using CorelDRAW X7 (https://www.coreldraw.com/en/pages/coreldraw-x7/).Full size imageThere are 33 rhomboidal caudal vertebrae that decrease in size gradually towards the posterior end of the tail. Caudal vertebrae CV13–21 have strap-shaped neural spines. Caudal ribs are present in CV1–11. They are flat, arch-shaped bones directed slightly posteriorly. The size of the ribs remains roughly the same from CR1–5, but this decreases suddenly from CR6–11 (Fig. 4c). The distal ends of CR3–8 are flat, while more posterior ribs have pointed ends.Figure 4Selected postcranial parts of WIGM SPC V 1105. (a) gastralia near the sacral region in ventral view, the arrow indicating each gastralium consists of one medial element and four lateral elements; (b) sacral region in dorsal view; (c) part of the caudal region in ventral view. cr5 caudal rib 5, cv5 caudal vertebra 5, cv15 caudal vertebra 15, dr19 dorsal rib 19, dv16 dorsal vertebra 16, dv17 dorsal vertebra 17, dv19 dorsal vertebra 19, il ilium, pu pubis, sr1 sacral rib 1, sr2 sacral rib 2, sr3 sacral rib 3, sv1 sacral vertebra 1, sv2 sacral vertebra 2, sv3 sacral vertebra 3. Scale bar = 5 cm. The figure is generated using CorelDRAW X7 (https://www.coreldraw.com/en/pages/coreldraw-x7/).Full size imagePectoral girdle and forelimbThe pectoral girdle is exposed in ventral view (Fig. 3a,b). The interclavicle is an arrowhead-shaped bone with a strongly concave posterior border and two posterolaterally directed lateral processes, unlike the more diamond shape of D. gracilis24. Its tip points anteriorly but does not reach the anterior margin of the pectoral girdle between the clavicles. The clavicle is an L-shaped, strap-like bone with a characteristic prominence anterolaterally, as in D. acutidentatus and larger than in D. gracilis17,24. The clavicle develops a tiny posterolateral process, overlying the dorsal surface of the scapula. The tapering medial process expands to meet its counterpart, forming the anterior margin of the pectoral girdle. The scapula is exposed in ventral view, so the dorsal blade is covered. In this view it is sub-rectangular, with a rounded anterior margin and two posterior facets for the clavicle and humerus, angled obliquely and separated by a small ridge. The coracoid is a strap-shaped bone with proximal and distal ends widened, and the largest element in the pectoral girdle. Its anteromedial margin is more strongly concave than the posteromedial margin. Proximally, the coracoid is flattened and meets the contralateral element in a straight median facet. Distally the coracoid is more robust and expanded anteriorly into a broad rounded process on the anterior margin. The distal margin is straight and articulates with the scapula anteriorly and has a smaller articulation with the humerus posteriorly on a smaller, triangular posterodistal process. There is a small foramen exposed near the anterodistal margin along the scapular facet, larger than in Dianmeisaurus gracilis24.Both forelimbs are nearly complete, ventrally exposed, about 13.7% of the body length (Fig. 3a,b). The humerus is strongly curved (40°) and shorter than the femur (Table 1). The proximal articular surface is rounded, with a larger facet for the scapula than the coracoid, while the articular surface of the distal end is convex, contacting the radius and the ulna with two straight, oblique facets. These facets are more strongly offset than in D. acutidentatus17. There is no evidence for an entepicondylar foramen20,24. The ulna and the radius are nearly equal in length and relatively gracile compared to the humerus (Table 1). The two ends of the ulna are equally widened, while the ends of the radius expand less obviously and are directed slightly medially.There are more than four elements in the carpus, all round and flat in ventral view. The intermedium is slightly larger than the ulnare (Table 1), unlike in D. acutidentatus17, and articulates mediodistally to the ulna, medially to the ulnare. Distal carpal 2 is the largest of the distal carpals and articulates distally between the intermedium and ulnare. Distal carpals 3 and 4 are present but extremely reduced. The metacarpals are elongate and strongly hourglass shaped. Metacarpal 1 is the shortest of the five while metacarpals 2–4 are almost equal in length, and metacarpal 5 is slightly shorter. All the digits are directed towards the ulnar side of the limb. The interosseous space between metacarpals 4 and 5 is the widest. The phalangeal elements are well preserved, but digit 5 of the right manus demonstrates unusual preservation, which will be discussed further in the Discussion. The ungual phalanges of digits 4 and 5 on the left are small and round, while the ungual phalanx of digit 5 on the right is missing. Given that, the forelimb is likely to have had a phalangeal formula of 2–3–4–4–3.Pelvic girdle and hindlimbThe pelvic girdle is exposed ventrally (Fig. 3c,d). The pubis is a large plate-like bone. Both the anterior and posterior margins of the bone are concave near the distal end (about one-third of the whole length), forming a ‘waisted’ shape that is narrower than in Dianmeisaurus gracilis24. The ischium is large and irregularly shaped. Medially it is expanded into a large, squared, plate-like portion that meets the contralateral element along a straight median symphysis. Anterodistally, the ischium is waisted, separating the large, robust anterodistal process with a broad, rounded end that contacts the distal pubis and ilium to form the acetabulum. The anterodistal process is narrower and more strongly offset from the main body than in Dianmeisaurus gracilis24. Posterodistally there is a further broad extension. The thyroid fenestra is large and rectangular and is bounded by the posterior pubis and anterior ischium on both sides. The ilium is covered by the pubis and the ischium in ventral view.The left hindlimb is well preserved and exposed in ventral view (Fig. 3c,d), and the amputated right femur is discussed below. The femur is long and rounded with a slightly waisted epiphysis; it is larger and slenderer than the humerus (Table 1). The proximal end is wider than the distal but is damaged in this specimen. The tibia and the fibula are similarly elongate bones, with the tibia somewhat more robust but more similar in size than in the holotype of D. acutidentatus17. Both have slightly expanded proximal and distal ends, but the proximal end of the fibula is hidden beneath the distal femur. The stronger waist on the fibula gives it a more strongly curved appearance and creates a large interosseous fenestra.The astragalus and calcaneum are the only elements of the tarsus. The astragalus is larger than the calcaneum and located between the distal tibia and fibula with a pointed proximal margin (Table 1). The facets of the astragalus contacting the tibia and the fibula are straight. The calcaneum is subcircular. Length increases from metatarsals 1–4, then decreases in metatarsal 5; metatarsal 1 is the shortest. All the metatarsals have an elongate hourglass shape. The pes is not so well preserved, as digits 1 and 2 are crushed together. The phalanges are less elongate than the metatarsals and shaped like waisted cylinders, except for the ungual phalanx of digit 5; consequently, there may be some missing ungual phalanges from the other digits. The pedal phalangeal formula cannot be determined due to the preservation.Phylogenetic analysisWe added WIGM SPC V 1105 to the cladistic matrix of Lin et al.27 and replicated their analytical methods in PAUP* version 4a169. Our cladistic analysis produced four most parsimonious trees (tree length = 485 steps, CI 0.388, RI 0.622). Strict consensus of these trees (Fig. 5) matches the result of former studies, in that Diandongosaurus share a close relationship with Dianmeisaurus24.Figure 5Strict consensus tree of four most parsimonious tree (TL = 485 steps, CI = 0.388, RI = 0.622), demonstrating the phylogenetic position of WIGM SPC V 1105. Bootstrap support values ≥ 50% (1000 replicates) are labelled. The figure is generated using Adobe Illustrator 2021 (https://www.adobe.com/products/illustrator.html).Full size imageDiandongosaurus shows some similarities with Keichousaurus and Dianopachysaurus18,19, but many morphological differences exist. Keichousaurus and Dianopachysaurus have small tapering teeth19,20, while Diandongosaurus has serried long fang-shaped teeth. The supratemporal fenestra of Diandongosaurus is oval-shaped and larger than in the other two taxa considering the size of the orbit. The caudal ribs of Dianopachysaurus develop a tapering distal end, different from Diandongosaurus, whose caudal ribs have a flat distal end17,20.Diandongosaurus also differs from other Triassic eosauropterygians. The strongly procumbent anterior teeth discriminate it from the pistosauroids, which have upright anterior teeth. The size of the supratemporal fenestra is noticeably larger than in Qianxisaurus28, while the characteristic tapering snout of Wumengosaurus29 differs from the blunt snout of Diandongosaurus. Its clavicle develops an anterior process, which does not exist in European pachypleurosaurs. Diandongosaurus has a smaller supratemporal fenestra than in Lariosaurus and Nothosaurus, in some species of which it is nearly twice the size of the orbit.WIGM SPC V 1105 broadly resembles D. acutidentatus but differs in several features, including being considerably larger and the constricted snout of WIGM SPC V 1105 is a novelty in pachypleurosaur. These morphological distinctions between WIGM SPC V 1105 and D. acutidentatus could be regarded as evidence for establishing a new species. Alternatively, WIGM SPC V 1105 lacks the pterygoid opening in the two referred specimens (specimen NMNS-000933-F03498 and BGPDB-R0001) of D. acutidentatus18,23, and other differences, like the larger size and the rounded ends of humerus and femur, could have been caused by ontogenetic variation or even preservational issues. Based on previous documented specimens, interspecific variation of phalangeal formula exists in D. acutidentatus, as the pedal formular counts 2–3–4–5–4 in the holotype, but 2–3–4–6–4 in the referred specimen BGPDB-R000123. In this case, WIGM SPC V 1105 could be an adult of D. acutidentatus. Given these considerations, we assigned WIGM SPC V 1105 as a conformis (cf.) of D. acutidentatus. More

Oocyte size frequency distributionThe OSFD, based on wholemount analysis (formalin-preserved diameter measurements), did not show any hiatus between the assumingly largest PVOs and the smallest VO (Supplementary, Fig. S1). The corresponding mean threshold value, determined statistically by the Gamma/Gaussian method (see technical details below), was 192 µm (95% CI: 187–196 µm) (Supplementary, Fig. S1). Based on histology, this value was, however, at ~ 230 µm, i.e. the formalin-preserved oocyte diameter of PVO4c (Supplementary, Figs. S2B, S3, Table S1).Spawning progressAddressing firstly “the population (wholemount) data set” of 1561 individuals (Table S2), the relative frequency of early-spawning (ORC1), mid-spawning (ORC2), and late-spawning (ORC3) females changed significantly as the spawning season progressed, although with dissimilarity between 2018 and 2019 (Supplementary, Fig. S4). Overall, a significant difference was found among the ORCs frequencies between the two field-sampling years (two-way ANOVA; p = 0.003). In June 2018, over 60% of the females caught were very late spawners or spent (ORC4), this relative frequency increased to almost 90% in July 2018 (Supplementary, Fig. S4A). For 2019, the ORC4 in June was about 50% (Supplementary, Fig. S4B). Combining these 2018 and 2019 data sets, the subsequent comparison showed that July 2018 clearly differed in terms of ORC (a posteriori Tukey test; Supplementary, Fig. S5). More females in mid-spawning were recorded in May and June 2019 compared to the same months in 2018, though this noted difference was statistically insignificant (Supplementary, Fig. S5). Altogether, these outlined variations in ORC (Fig. 1) may be related to survey coverage, i.e. in 2018 these samples were collected in Nordic waters, while in 2019 exclusively within the main spawning area (Fig. 2).Figure 1Wholemount counts of previtellogenic (PVO) versus developing oocytes (VO and FOM) used within the ultrametric method to categorize the “stage of spawning” represented by the oocyte ratio category (ORC). The resulting ORC category (ORC1-4) is showed above each panel. VOs includes cortical alveoli oocytes.Full size imageFigure 2Map with location and number of all mackerel female samples collected from May 2018 to June 2019. The map was created using R v4.0.4 (https://www.r-project.org/) (see details at “Material and methods” section).Full size imagePopulation-level ORC and biometrics appeared linked, the latter represented either by total length (TL)-based gonadosomatic index (GSITL) or relative condition (Kn) (Fig. 3). The 2018 results showed that Kn was higher (p  More

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