Contributions to Zoology, 81 (1) – 2012Valerio Scali; Liliana Milani; Marco Passamonti: Revision of the stick insect genus Leptynia: description of new taxa, speciation mechanism and phylogeography

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Quite clearly, mitochondrial DNA genes had demonstrated that all European species of Bacillus and Clonopsis (Areolatae) and Leptynia (Anareolatae) pertain to a same monophyletic clade (Galassi, 2002); therefore the commonly accepted suborder splitting into Areolatae (represented Bacillus and Clonopsis) and Anareolatae (now represented by Leptynia and Pijnackeria) must be questioned; in this context the occurrence of the tiny area apicalis among the Phasmida (= Phasmatodea) should be considered a homoplasious (or convergent) character state, although it has been also envisaged as a homologous plesiomorphic trait (Bradler, 2009). At any rate, while further evaluation is pending, we can keep the attribution of Leptynia to the family Diapheromeridae, subfamily Pachymorphinae, tribe Gratidiini (Otte and Brock, 2005), since, after all, all morphological and structural body traits of Leptynia are coherent with the general features of those systematic groupings (see Appendix).

The karyotypes neatly separate all species, but are not diagnostic among the three L. attenuata subspecies (Fig. 3). It must be noted, however, that while to the same number reported for L. attenuata subspecies practically indistinguishable karyotypes correspond, in L. caprai and L. annaepaulae to the same score of 39/40 chromosomes consistently different karyotypes occur (Fig. 3). In this connection we want to mention that stable karyotypes have been always obtained from all analyzed specimens within each species; with one exception, however, since the karyotype of Viso del Marques specimens (Calatrava Region, southern part of L. caprai range) was different from the other L. caprai populations, and surprisingly more similar, although not identical, to that of L. annaepaulae by having a submetacentric X chromosome - the largest in the karyotype - and also possessing the second large metacentric pair just like L. annaepaulae, both lacking from L. caprai, whose sex chromosomes and large pairs are all acrocentrics or subacrocentrics (Fig. 3). This finding is to be confronted with the observation that the linearized NJ cox2-based tree (see Passamonti et al., 2004) put these specimens within the L. caprai cluster. The karyotype of L. attenuata attenuata is characterized by a large submetacentric X, and a small acrocentric Y (1st pair). Additional outstanding pairs are the metacentric 2nd, and the acrocentric 4th with satellites. This karyotype, with only minor differences, is also found in L. attenuata iberica and L. attenuata algarvica.


Fig. 3. A) Karyotype of L. caprai female: 2n = 40, XX/X0. Note the acrocentric/subacrocentric structure of all large pairs, including the X chromosomes (the largest). B) Karyotype of L. annaepaulae male: 2n = 40, XX/X0. Note the submetacentric X, the 2nd metacentric pair and the 13th and 18th pairs with satellites. C) Karyotype of L. attenuata attenuata male, 2n = 36, XX/XY, with a large submetacentric X, and a small acrocentric Y (1st pair). D) Karyotype of L. caprai found at Viso del Marquès: an intermediate situation between L. caprai and L. annaepaulae can be appreciated; furthermore a re-patterned chromosome (# 10) can be observed. In brackets is reported the same re-patterned chromosome taken from another plate to support its maintenance in germ cells.

The Bayesian tree based on cox2 mitochondrial gene (Fig. 2) showed a clear splitting between L. annaepaulae and all other Leptynia samples (pp = 1.00). The remaining Leptynia are joined by a vast unresolved polytomy: L. attenuata is not retrieved as a clade, possibly due to the low variability of the cox2 gene, but also L. caprai was not recovered as a single clade; the issue should be further investigated. However some groups can be recognized: L. montana (pp = 0.99), L. attenuata algarvica (pp = 1.00), and L. attenuata iberica (pp = 1.00); on the other hand, since the L. attenuata attenuata clade was supported in previous analyses through Maximum Parsimony and Maximum Likelihood, based on the very same sequences (see Passamonti et al., 2004), we think we are allowed to maintain this node (here marked with an asterisk). Moreover, Fig. 4 highlights the substitutions observed in the same sequenced haplotypes of cox2 gene: all Leptynia taxa, with the noteworthy exception of L. caprai, show diagnostic mutations; on the other hand, L. caprai shares several mutations with the other taxa.

On the basis of karyotype features and genetic differentiation, a corresponding morphological analysis has been carried out, by investigating most of the commonly recorded parameters of bodies and eggs (see Scali, 1996; Scali and Milani, 2009) (Table 2).


Fig. 4. Substitutions in the sequenced cox2 partial gene of Leptynia. Private mutations marked in grey. Sample acronyms as in Fig. 2. All taxa, excluding L. caprai, show diagnostic mutations. L. caprai shares several mutations with the other taxa.


Table 2. Chromosome numbers and main body and egg parameter ranges of Leptynia taxa. Lengths are given in mm and angles in degrees. Body measures do not include antennae and cerci. The number of measured specimens are given in brackets: for L. caprai, L. montana and L. attenuata algarvica it could also include, when appropriate, those cumulatively reported in Scali (1996), amounting to about 40 for each taxon; abdom. terg. = abdominal tergite.

Also the new Leptynia taxa are small phasmids - around 40mm the males and 50mm the females - with the same slender body appearance as that observed in co-generic species; the common sexual dimorphism is always apparent, with males much thinner than females (Figs 5 and 6).


Fig. 5. Holotype (male) of Leptynia annaepaulae sp. n. Note the widespread brown pigmentation of the tergites, the sharp white line above the pleura and the canella colour of sternites. It is also possible to just see the relatively long antennae (lozenge), particularly if compared with those of the female (Fig. 6; see also Fig. 7), the profile of the sub-genital operculum (arrow) and the terminal claspers (arrowhead). Bar = 1 cm.


Fig. 6. Paratype (female) of Leptynia annaepaulae sp. n. Note the pink nuance of the whole body colour, the bright white lateral line, the short antennae (lozenge; see also Fig. 7) and the straight projecting cerci (arrowhead). Bar = 1 cm.

From an overview of the reported metric and meristic traits it can be seen that in the males the 10th:9th ratio value appears to be fully diagnostic for L. annaepaulae, whereas no other sharp distinguishing trait exists to separate a given taxon from all others; rather, only morphological or dimensional trends could be observed for the analyzed traits, since the recorded features or values always suffer some overlapping (Table 2). However, additional, mainly qualitative characters - such as the subanal vomer features and cercus tooth shape/size - although rather tricky, allowed a reasonably safe diagnosis of a few Leptynia taxa (Table 3 and Appendix).

L. attenuata algarvica males are the smallest, while those of the other two L. attenuata subspecies largely overlap each other and with the remaining species. L. attenuata algarvica females too tend to be small, but their size range amply overlaps or even lays within those of the remaining taxa. The article number of antennae is the same in males and females of all species; however, their length is consistently higher in males than females, thus confirming the well known sexual allomorphism for this character in stick insects (Fig. 7); the very low values of antennal lengths recorded for L. attenuata algarvica females could be linked to their small body size, but also the reduced sample could be responsible for this finding.


Fig. 7. A) Antennae of a Leptynia male, with 17 articles: all taxa share the same antennal structure and have a similar length range with higher values than females (sex allomorphism). Bar represents 1 mm. B) Antennae of a Leptynia female, with 16 articles. The structural antennal plan is the same as that of males, but the single articles are shorter. Incomplete or additional segmentations can modify the basic article number in both sexes. Legend: arrow - scapus; arrowhead - pedicellus. Bar = 0.8 mm.

One additional trait often utilized to characterize related orthopteroid taxa, independently from absolute specimens’ size, is the hind femur reach when positioned parallel to the abdomen. In tested males of the already described species, the hind leg femur invariably attained about the half of the 7th abdominal segment, thus being of no diagnostic value. The same parameter gave similar results for L. attenuata and L. annaepaulae males; the only finding at variance has been obtained from L. attenuata iberica: out of six measured males two had proportionately longer legs, since their femur reached almost half of the 8th abdominal segment. A slightly more significant result has been obtained from females: the hind femur reach has been observed to increase from L. attenuata algarvica (half of the 5th segment) to L. attenuata iberica (from 5th to 6th/7th segment joint) and, further, to L. attenuata attenuata (from the base of 7th segment to half of it). In L. annaepaulae females the hind femur constantly reaches from half of 6th up to 7th segment articulation.

As already pointed out, the presence of a subanal vomer (about 1mm long) is a major diagnostic character of Leptynia males, when compared to those of the related Pijnackeria genus, which lack it (Scali, 2009a, 2009b). In addition, its shape has now turned out to realize different patterns among Leptynia species and even subspecies. As a matter of fact, the proximal part the vomer base is swollen, almost spherical, in most specimens of L. attenuata attenuata and L. attenuata algarvica; moderately swollen and only slightly wider in L. attenuata iberica, flat and decidedly wider than its following segment in L. annaepaulae, where it also shows a lobate structure (Fig. 8A, C, E). Furthermore, in many L. attenuata males the vomer may appear furrowed on the base and/or the distal, narrower part of it (Fig. 8C, E). The same observations revealed that even for the already known L. attenuata and L. caprai differences can be perceived, so that a complete taxon-specific series could be obtained (Table 3).


Fig. 8. Sample of male and female terminalia of Leptynia, showing some significant structures. A-B) L. attenuata attenuata male: the swollen smooth subanal vomer base (1) its furrowed distal segment and the cercus (2) with a slender tooth (arrowhead); the tapered operculum (3). C-D) L. attenuata algarvica male: the vomer swollen base, the stout tooth (arrowhead) and the truncated operculum (3). E) L. annaepaulae male: the flat and lobed appearance of the vomer base (1); the short, pointed tooth (arrowhead); the posterior operculum border (3). F) L. attenuata iberica male: the low conical tooth (arrowhead), with a wide base. G) Female terminalia of L. attenuata attenuata showing the 1st ovipositor valve (6) the tapered, soft 10th sternite (7) and straight cerci (2). Legend: 1 - vomer; 2 - cercus; 3 - male operculum; 4 - 9th abdominal tergite; 5 - 10th abdominal tergite; 6 - ovipositor valve (8th abdominal sternum); 7 - 10th sternite; arrowhead - cercus tooth. Scale bars: A, B, C, E, F = 0.5 mm; D = 0.2 mm; G = 1 mm.


Table 3. Comparisons between subanal vomer and cercus traits in Leptynia males (see Fig. 8).

Male cerci are well developed and in the adults assume a typical conformation of claspers; a peculiar trait of Leptynia claspers is the occurrence of a projecting tooth of variable size and shape, placed near the base of each cercus. When such teeth were compared among the first described species (L. caprai, L. montana and L. attenuata), species-specific patterns could be appreciated: it appeared very low with a wide base in L. caprai, longer and thick in L. montana, longest and stout in L. attenuata, so that tooth features appeared fully diagnostic (Scali, 1996). The new taxa as well show differing cercus tooth traits: slender, short and pointed in L. annaepaulae; slightly longer, thin with a blunt apex and obliquely inserted in L. attenuata attenuata; low, conical with a wider base in L. attenuata iberica; long and more robust in L. attenuata algarvica (Fig. 8B, D-F).

Therefore, the clear cut differences observed among the first described species have become rather subtle and careful comparisons are now needed to make a sound assessment of each taxon.

A partially diagnostic condition is found when egg size and chorion features are compared. All Leptynia species lay thin, elongated eggs, but L. caprai and L. annaepaulae appear to have comparably longer eggs than others (Table 2); accordingly L. montana and L. attenuata lay shorter eggs, amply overlapping each other for size range. The higher length values of L. caprai and L. annaepaulae are mainly due to their shared traits of developing both a raised polar mound and a dome-shaped operculum, while in the shortest eggs a shallow polar mound and a flat operculum are realized (Fig. 9). All this is mirrored in the width to length ratios: owing to the almost invariant egg width values in all taxa, the lowest ratio figures are found for L. caprai and L. annaepaulae, while for the remaining clearly higher values are obtained (Table 2). At the same time it can be seen that L. annaepaulae eggs, together with L. caprai, show the highest values of positive opercular angle; the value is intermediate in L. montana and variable within L. attenuata, where in the nominal subspecies it can be neutral or even become negative (Fig. 9B-E). On the whole, the series of egg data sets heavily blurs the picture resulting from the early description of Leptynia species, where the opercular angles appeared to constitute fully diagnostic traits (Scali, 1996). The fine chorionic pattern on both egg capsule and the lance-tip shaped micropylar area is similar in the new taxa and is made by a carpet of irregularly shaped pin-heads (about 2µm in diameter), with a superimposed irregular pattern of sparse ribbons. These attain a maximum development and density on the roundish operculum and the polar mound, particularly in L. annaepaulae, where they may take the appearance of projecting cristae as it also occurs in L. caprai (Fig. 9).


Fig. 9. Eggs of Iberian phasmids. A) Dorsal view of egg capsule of Pijnackeria, showing its outstanding ribbon pattern, the micropylar area (asterisk), micropylar cup (arrowhead) and opercular opening (arrow). B) Dorsal view of the slender egg of L. attenuata attenuata with much lower ribbon-net developmentand a flat operculum (arrow). C) Dorsal view of the very elongated capsule of L. caprai, showing, the micropylar area (asterisk), the raised operculum (arrow) and the developed polar mound at the opposite pole. D) Side view of L. annaepaulae eggswith the positive angle of the opercular opening (right) and the raised polar mound (left). E) anterior part of L. attenuata attenuata egg with small pin’s heads and an almost perpendicular operculum. F) The shallow operculum of L. attenuata algarvica. G) The dome-shaped one realized in L. caprai and L. annaepaulae. Scale bars: 1 mm in A-D and 0.1 mm in E-G. Legend: arrow, operculum; arrowhead, micropylar cup; asterisk, micropylar plate.

Detailed descriptions of the new taxa and their comparative diagnosis, are given in the Appendix (Tables 4 and 5).