New fossil stoneflies (Plecoptera: Arctoperlaria) from Australia testify ancient dispersal across Pangea

The stonefly suborders Arctoperlaria and Antarctoperlaria reflect the current division of the diversity of this insect order between the Northern and Southern Hemispheres. However, there are several exceptions to this pattern, the most notable being the family Notonemouridae, which is phylogenetically deeply subordinate within the northern Arctoperlaria, but distributed in South Africa, South America, and Australia. Various hypotheses have been proposed regarding the circumstances of their dispersal to the south. Some estimated their origin as relatively recent, with long-distance dispersal to the southern continents in the Late Cretaceous or early Paleogene. On the other hand, fossils of Notonemouridae have been dated to the Middle Jurassic, proving the lineage is very ancient. However, all known notonemourid fossils originate from Asia and the timing of their dispersal to the south cannot be precisely estimated. Here we report new fossil stoneflies from the Late Jurassic Talbragar Fish Beds, Australia, described as Talbragaria australis gen. et sp. nov. and attributed to Notonemouridae. This finding represents the first fossil evidence of the northern suborder Arctoperlaria in the Southern Hemisphere, and confirms the north-to-south dispersal of Notonemouridae across Pangea prior to the continental break-up.


Introduction
Plecoptera have an unusual antitropical distribution, being much more speciose in high latitude environments than in tropical regions (Zwick 2000).The current higher-level classification of the order also reflects this division, as is implied by the names of two suborders, Antarctoperlaria and Arctoperlaria.These two major clades are widely agreed upon and strongly supported as monophyletic by several approaches (Zwick 2000;Ding et al. 2019).Antarctoperlaria are restricted to the Southern Hemisphere, whereas Arctoperlaria are restricted to the Northern Hemisphere, albeit with a few exceptions.
Those exceptions are represented by three lineages of originally Laurasian Arctoperlaria found in the Southern Hemisphere, namely the genus Neoperla (Perlidae) in Africa, subfamily Acroneuriinae (Perlidae) in South America, and family Notonemouridae, which shows a disjunct South African (included Madagascar), South American, and Australian (including New Zealand) distribution (Zwick 2000).The timing and circumstances under which these three lineages reached the Southern Hemisphere differ.The originally Asian genus Neoperla probably reached Africa relatively recently during the Paleogene (Zwick 2000).For South American Acroneuriinae, Stark and Gaufin (1976) hypothesized that recent representatives arose from a now extinct Asian or European group which dispersed across Northern Africa in the Cretaceous and became established in South America.This hypothesis is partly supported by the presence of Acroneuriinae in Late Cretaceous Burmese amber (Sroka et al. 2018).
In the case of Notonemouridae, conflicting hypotheses have been proposed.Phylogenetically, Notonemouridae is a sister lineage to Laurasian Nemouridae, nested within Euholognatha (Zwick 2000;Ding et al. 2019), and despite their current distribution range being restricted to the Southern Hemisphere they are certainly of Laurasian origin (Illies 1965;Zwick 2000;Ding et al. 2019).Some authors have considered that the present distribution of notonemourids is attributable to Gondwanan vicariance (Illies 1965;Zwick 2000) and results from dispersal across Antarctica (Zwick 1981(Zwick , 1990).On the contrary, McCulloch et al. (2016) estimated the age of Notonemouridae as postdating the continental break-up and hypothesized a later colonization of Gondwana by Notonemouridae by long-distance dispersal.Similar results were presented by Ding et al. (2019), who dated the divergence of Notonemouridae at 71 Ma, suggesting that the ancestors of this family spread to the Southern Hemisphere during the Late Cretaceous.An important piece of fossil evidence was presented by Liu et al. (2011), who described the family Pronemouridae, supposedly representing the stem group of Notonemouridae + Nemouridae clade, occurring already in the Middle Jurassic of China.Based on this finding, Liu et al. (2011) suggested the dispersal of Notonemouridae to the Southern Hemisphere took place by at least the Early Cretaceous.Other fossil evidence for the antiquity of Notonemouridae was brought forward by Cui et al. (2018), who attributed Paranotonemoura zwicki Cui and Béthoux, 2018, also from the Middle Jurassic of China, to the crown group Notonemouridae.The same authors also transferred another species to Notonemouridae, Perlariopsis fidelis Sinitshenkova, 1987, described earlier from the same epoch of Mongolia and originally attributed to the family Perlariopseidae by Sinitshenkova (1987).With the discovery of Notonemouridae in the Jurassic of China and Mongolia, Cui et al. (2018) proved that the age of this family is much older than hypothesized by other authors.Nevertheless, they did not prove that the dispersal of this lineage to the Southern Hemisphere is also ancient, since their fossils originated from Asia.Cui et al. (2018) nevertheless assumed that the north-to-south migration of Notonemouridae took place between 220 and 160 Ma.
Therefore, the colonization of Southern Hemisphere by Notonemouridae might have been possible prior to the main continental break-up.This scenario seems to be more plausible than a later long-distance dispersal overseas given the restricted flight ability and short adult lifespan of stoneflies (Zwick 1980).Nevertheless, there has been no fossil evidence of Notonemouridae being present in the Southern Hemisphere.Therefore, the time of dispersal of this lineage to the south could not be confirmed.
We report new Late Jurassic stonefly fossils from the Talbragar Fish Beds in Australia, which we attribute to Notonemouridae.Our findings represent the only fossil evidence of Arctoperlaria in the Southern Hemisphere in deep time, and therefore are of considerable value for understanding the dispersal of stoneflies to Gondwana and the historical biogeography of this insect order in general.

Locality
The material originates from the Talbragar Fossil Fish Beds, situated north of Gulgong in central New South Wales, Australia.A recent analysis of zircon crystals from this strata dates it to the very late Oxfordian-Tithonian (Late Jurassic), ca 157.3-145.0Ma (Turner et al. 2009).The deposit represents a shallow-water-lake palaeoenvironment at the northern end of the deposit, grading to a shoreline palaeoenvironment towards the southern end of the deposit.It was part of a productive lake supporting a large population of fish and a diverse aquatic/shoreline and terrestrial woodland fringe palaeoecosystem, which has produced a significant number and variety of insect fossils (Beattie and Avery 2012).Several new insect taxa have recently been described, comprising members of the orders Odonata (Beattie and Nel 2012), Hemiptera (Chen et al. 2019;Li et al. 2022), Coleoptera (Fikáček et al. 2014;Ashman et al. 2015;Oberprieler et al. 2016), Hymenoptera (Oberprieler et al. 2012), and Diptera (Oberprieler and Yeates 2012, 2014;Oberprieler et al. 2015).

Material processing
The material was examined dry and under a film of ethyl alcohol using Olympus SZX7 and Leica M205 C stereomicroscopes.The photographs were taken using a Canon EOS 550D and a Canon EOS 1200D digital camera equipped with MP-E 65 mm and EF-S 60 mm macro-lenses or attached to the Leica M205 C stereomicroscope.Original photographs were processed by stacking and editing using Adobe Photoshop™ version CS6 (Adobe Systems Incorporated, San Jose, U.S.A.).The measurements of individual body parts were inferred from the photographs taken with a calibration scale.
All the material is deposited in the Australian Museum, Sydney, Australia under the accession numbers as specified below.Diagnosis.By monotypy, as for the type species.

Results: systematic palae ontology
Type species.Talbragaria australis sp.nov.by present description.
Forewing venation only partially preserved (Fig. 1A, B,   2F,G) and F.137 324 (Fig. 2H).Due to their state of preservation and lack of diagnostic characters, it is not possible to attribute them unambiguously to T. australis gen.et sp.nov., although due to their similarity in size they might be conspecific.

Phylogenetic placement
Although the wing venation is not completely preserved in the specimens of Talbragaria australis gen.et sp.nov., the anterior part of the wings is unambiguously discernible (Fig. 1A, B, F-H).It features the so-called nemourid X, consisting of ScP that fuses with RA and again diverges from it more distally (Cui et al. 2018), the basal part of RP bifurcation and a crossvein between RP and M. According to Zwick (2000), this pattern of veins is generally present in the families Nemouridae and Notonemouridae (Arctoperlaria, Euholognatha).Although the nemourid X indicates a close relationship between T. australis sp.nov.and the Nemouridae + Notonemouridae clade, a similar pattern of veins can also be observed in the likewise euholognathan families Taeniopterygidae and Capniidae (Zwick 2000).However, the possible attribution of T. australis sp.nov. to these families can be excluded using other characters.Capniidae are characterised by an apomorphic reduction of m-cua crossveins (at most two) and cua-cup crossveins (at most one) (Zwick 2000).In our fossils, the number of respective crossveins is obviously higher, despite the fragmentary preservation, but there are no crossveins in the apical portion of the wings (Fig. 1F).Taeniopterygidae exhibit an elongated second tarsomere, which is at least 4/5 of the length of the third tarsomere and represents an apomorphy of this family (Nelson 2009).The second tarsomere in T. australis sp.nov. is shorter, subequal to 2/3 of the length of the third tarsomere (Fig. 1C).Since the first tarsomere is missing in available specimens of T. australis sp.nov., there is a possibility that the second tarsomere is broken basally and incomplete.However, since the first tarsomere is not preserved on the counterpart, we assume that the second and third tarsomeres are the only ones present in the fossil and the fragmentation of the tarsus happened not during the splitting of the matrix, but early in the fossilization process.Thus, a disarticulation between tarsomeres is more probable than a crack within a basal part of tarsomere.Consequently, we consider the second tarsomere to be likely complete.Therefore, all available morphological evidence testifies for the placement of T. australis sp.nov.into either Notonemouridae or Nemouridae.Zwick (2000) questioned the monophyly of notonemourids based on the presence of an ovipositor in some genera only.Based on morphology, he considered Notonemouridae as possibly representing a paraphyletic assembly (grade) of early nemourid lines surviving on fragmented Gondwanaland.However, the families Notonemouridae and Nemouridae were recovered as sister groups in the molecular phylogeny of Ding et al. (2019) and the monophyly of Notonemouridae is strongly supported by McCulloch et al. (2016).One more purely fossil family with a close relationship to the Nemouridae + Notonemouridae clade is represented by Pronemouridae, hypothesised by Liu et al. (2011) to represent the stem group of this clade.
The only one of these families known from the Southern Hemisphere is Notonemouridae (Zwick 2000); therefore its presence in the Upper Jurassic of Australia is more probable than in the case of Nemouridae or Pronemouridae.Cui et al. (2018) introduced a new apomorphic wing venation character to define Notonemouridae, namely CuP in the forewing closely approaching the posterior wing margin (just distal of the end of AA1), then diverging from it, until it reaches it more distally.This character is only partially observable in our fossils, since the posterior portion of wings is not completely preserved.In the specimen F.136 857, a short visible portion of CuP is distally converging with CuA (Fig. 1G, H).It indicates the course of CuP as characteristic for Notonemouridae.Nevertheless, other evidence for the attribution of T. australis sp.nov. to Notonemouridae is represented by the structure of the female genitalia that very closely resembles some Recent notonemourids.It exhibits a pronounced corrugated subgenital plate posteriorly on sternite VIII enabling interlocking with the male epiproct or other copulatory structures, and posteriorly narrowed subanal plates on sternite IX (compare Figs 1D, E, 2D, E and fig.3.5 in Stevens 2008).Furthermore, T. austra-lis sp.nov.differs from the families Pronemouridae and Nemouridae in the structure of the tarsi.The second tarsomere in both above-mentioned families is distinctly short, reaching to approximately one-half of the third tarsomere (Nelson 2009;Liu et al. 2011).The length of the second tarsomere in T. australis sp.nov. is slightly longer, subequal to two thirds of the length of the third tarsomere, which represents a value reported for several representatives of Recent Notonemouridae (Nelson 2009).Pronemouridae are further characterised by two plesiomorphic characters, CuA ending with two branches and multisegmented cerci (Liu et al. 2011).Cerci in T. australis sp.nov.are one-segmented (Fig. 1D, E), an apomorphic character shared with Notonemouridae and Nemouridae (Cui et al. 2018).The course of CuA in T. australis sp.nov. is not observable distally of the M-CuA crossvein, however, no bifurcation is visible in the preserved portion of CuA (Fig. 1F).Also taking into account the biogeographic pattern of the discussed individual stonefly taxa, we consider the attribution of T. australis sp.nov. to Notonemouridae as the most probable.
Within Notonemouridae, Cui et al. ( 2018) established a subfamily Paranotonemourinae to accommodate two fossil species of the genus Paranotonemoura Cui and Béthoux, 2018.The species Talbragaria australis sp.nov. is not attributable to Paranotonemourinae since the mp-cua crossvein is not more than twice as long as the longest crossvein in the area between CuA and CuP (Fig. 1F), which does not comply with the diagnosis of Paranotonemourinae (Cui et al. 2018).Therefore, we attribute T. australis sp.nov. to the subfamily Notonemourinae within Notonemouridae.
There are 23 extant genera of Notonemouridae (De-Walt et al. 2023).It is difficult and in some cases impossible to compare T. australis sp.nov.with these genera, since some are defined based on characters not visible in T. australis sp.nov.(notably all characteristics of male genitalia).In any case, given the estimated ages of Plecoptera clades (Cui et al. 2016(Cui et al. , 2018)), it is not reasonable to expect a Recent genus being already present in the Jurassic.We would like to refrain from implying closer relationship to any particular Recent genus, which is not really grounded because of the lack of reliable characters.Recent representatives of this family occur only in the fragments of Gondwana, suggesting that the southward dispersal was followed by the extinction of Notonemouridae in the Northern Hemisphere.This extinction possibly occurred in the mid-Cretaceous, when a massive increase of the extinction rate was reported for Plecoptera (Jouault et al. 2022).

Biogeography
The fact that Notonemouridae were present in Australia in the Late Jurassic and at about the same time also occurred in China and Mongolia (Cui et al. 2018) further indicates that the lineage must have been very widespread, considering that the dispersal route by land between these sites spanned several of today's continents (Fig. 3).Importantly, it also provides evidence that the lineages within stonefly suborders up to the level of some families were already diversified in the Late Jurassic.Given the fact that Notonemouridae is not a basal lineage in the Plecoptera phylogeny (Zwick 2000;Ding et al. 2019), this indicates that the split between the suborders Antarctoperlaria and Arctoperlaria occurred much earlier than the Late Jurassic.Thus, some published age assessments for this split are certainly underestimated.McCulloch et al. (2016) dated the split between suborders as Early Cretaceous (121 Ma, with a confidence interval 143-109 Ma) and Ding et al. (2019) estimated the split of Antarctoperlaria vs Arctoperlaria as Early Jurassic (181.45Ma, although with a wide confidence interval spanning most of Mesozoic, 106-250 Ma).Probably a more realistic estimate for the major divergences within Plecoptera were given by Cui et al. (2016Cui et al. ( , 2018) ) based only on the study of the fossil species.They suggested that the Antarctoperlaria vs Arctoperlaria split occurred in the early Mesozoic or the late Palaeozoic (approximately 260-230 Ma).Interestingly, this timing would rule out the possibility that the split between stonefly suborders was a vicariant event resulting from the continental break-up between Gondwana and Laurasia 215 to 175 Ma, as traditionally hypothesised (Zwick 2000;Ding et al. 2019).Since some Recent families were already diversified in the Late Jurassic, the split must have occurred much earlier, predating the break-up.

Conclusions
The newly described fossil stonefly species, Talbragaria australis gen.et sp.nov.from the Upper Jurassic of Australia, is placed in the family Notonemouridae on the basis of several morphological characters, in particular the wing venation pattern.This family is phylogenetically nested within a suborder Arctoperlaria, which is almost exclusively Laurasian.Our results provide evidence for the presence of Notonemouridae in the Southern Hemisphere prior to the main continental break-up, confirming the earlier hypothesis of north-to-south dispersal of notonemourids across Pangea.
Our finding from the Southern Hemisphere complements the journey of Notonemouridae by confirming their presence in the territory of Gondwana already in the Late Jurassic, since Talbragar Fish Beds are dated as Oxfordian-Tithonian (ca 157.3-145.0Ma) and Notonemouridae must have already been established in the area of current Australia at that time.Their dispersal to the Southern Hemisphere thus predates the rifting of Pangaea, exactly as presumed by Liu et al. (2011) and Cui et al. (2018).Therefore, the hypothesis of McCulloch et al. (2016) and Ding et al. (2019) about the more recent colonisation of Gondwana by Notonemouridae by long-distance dispersal postdating the Gondwana break-up can be rejected.
F-H), ScP adhered to RA and again diverges from it more distally, RA simple, crossvein between RA and RP close to the point of connection of ScP with RA.Further two crossveins between RA and PR more basally.RP bifurcated just distally from the crossvein between RA and RP.Two crossveins between RP and M in proximal portion of wing, further oblique slightly sigmoidal crossvein between RP and M more distally, close to RP bifurcation.
Additional Plecoptera specimens.Apart from the holotype and paratype of Talbragaria australis gen.et sp.nov., two further specimens of Plecoptera from the same locality were identified in the collection of the Australian Museum, Sydney, Australia under accession numbers F.136 851 (part and counterpart, Fig.