Dominican amber net-winged beetles suggest stable paleoenvironment as a driver for conserved morphology in a paedomorphic lineage

Paedomorphosis is a heterochronic syndrome in which adult individuals display features of their immature forms. In beetles, this phenomenon occurs widely in the superfamily Elateroidea, including the net-winged beetles (Lycidae), and, due to the usual flightlessness of paedomorphic females, it is hypothesized to cause speciation rates higher than in non-paedomorphic lineages. However, some fossils of paedomorphic lycids do not support this with palaeobiological data. Discovery of new Lycidae fossils attributed to the West Indian extant paedomorphic genus Cessator Kazantsev in the Dominican amber also suggests morphological stasis within this genus in the Greater Antilles. We describe Cessator anachronicus Ferreira and Ivie, sp. nov. based on adult males, as well as the first ever recorded fossil net-winged beetle larva of the same genus. We propose that the relatively young age of the studied fossils combined with the stable conditions in the forest floor of the Greater Antilles through the last tens of million years could explain the exceptionally conserved morphology in the net-winged beetles affected by the paedomorphic syndrome.

Paedomorphosis is a heterochronic syndrome in which adult individuals display features of their immature forms 1,2 . In beetles (Coleoptera), the paedomorphic syndrome can be expressed as a mosaic of characteristics, which can include the reduction of sclerotization and/or loss of flight ability, miniaturization of morphological structures, the predominance of a K-reproductive strategy and enhanced fecundity of females [3][4][5][6][7] . In extreme cases, adult paedomorphic beetles can be completely larviform and fully resemble their larval stages 6,8 . Despite the conspicuousness of this syndrome in beetles, especially in the superfamily Elateroidea, which includes clickbeetles (Elateridae), fireflies (Lampyridae) and rail-road worms (Phengodidae) 3,4,6,7,[9][10][11][12][13][14] , the processes that generate these modifications and overall effects of paedomorphosis remain largely unknown and poorly studied.
One of the attributes that has been hypothesized for paedomorphic lineages is a tendency for rapid diversification and higher speciation rates [26][27][28] . In other animal groups, higher speciation rates have been correlated with higher morphological plasticity 29,30 . Fossils of the paedomorphic lineages could provide valuable palaeobiological data to investigate evolution of paedomorphosis and, in particular, to test the higher speciation rates hypotheses. In the Elateroidea fossil record, several taxa affected by the paedomorphic syndrome were found in various families [31][32][33] . In the described fossil record of Lycidae, currently comprised by 12 known species 20,34-37 , only three species were assigned to lineages that are known to have individuals affected by the paedomorphic syndrome, i.e., Electropteron avus Kazantsev, 2013 39 . Such records indicate that paedomorphosis has been occurring in this family at least since ca. 100 Mya. Our recent survey of collections of Dominican amber specimens revealed four undescribed specimens of Lycidae, including the first lycid larva that could be identified to generic level. A study of this material and comparison with previously described taxa allowed the placement of these undescribed taxa in the extant paedomorphic Greater Antillean genus Cessator Kazantsev, 2009 (Leptolycini). Since Cessator is a well established paedomorphic lineage 38,40 , it can be predicted that in this poorly dispersing beetles affected by the paedomorphic syndrome, several million years old fossils would be morphologically rather distinct from extant congeners due to the increased speciation rate 41 . However, so far this does not seem to be the case found in the paedomorphic net-winged beetles from Dominican Amber 35,38,42 . So far, known Dominican amber lycid fossils challenge the hypothesis that lineages with presumed higher speciation and diversification rates should be correlated with an increased morphological plasticity over time, and the discovery of additional specimens is particularly intriguing. With all those controversies in mind, we not only provide descriptions of these newly found fossils but also propose potential explanations of the observed conserved and little changed morphology in paedomorphic lycids in the Greater Antilles from the time of the Dominican amber formation till present.

Remarks
Cessator is a West Indian genus of Lycidae currently known by one extant and two extinct species, including the one described here. The extant species, Cessator luquillonis Kazantsev, 2009, is known from the eastern part of Puerto Rico 40 , while the two fossils are from Dominican amber deposits. The diagnostic characters of the genus were given in Kazantsev 40 and Ferreira and Ivie 38 and will be expanded by Ferreira and Ivie 43 in their review of the Puerto Rican Leptolycini study, where many other species and the immature forms of the group are going to be reported. Etymology. Anachronism is defined by the Cambridge dictionary as a person, thing or idea that exists out of its time in history. The species name was given in allusion to its remarkable similarity with extant congenerics, even though being set apart by millions of years. The specific epithet comes from the word anachronous, with its Greek roots meaning ἀνά ana, 'against' , and χρόνος, khronos, 'time' , which in its adjectivized form changes to anachronicus.

Differential diagnosis. Cessator anachronicus sp. nov. can be distinguished from all other known fossil
Leptolycini by its color combination, having the thorax, proximal region of the humeri, and apical half of antennomere X and antennomere XI yellow ( Fig. 2A

Description.
Male. Possessing all generic characteristics described in Kazantsev 40 and Ferreira and Ivie 43 .

Discussion
Fossil Lycidae are known from the Cretaceous and Tertiary deposits from various parts of the world 36 . The only known impression fossil is a poorly preserved specimen from the Eocene Florissant beds in North America 34 . However, its identity is too uncertain to be considered in our discussion.  www.nature.com/scientificreports/ Tihelka, Huang and Cai, 2021 from the Cretaceous, and Protolycus Kazantsev, 2019 from Eocene), whilst all other species were described in genera for which one or more extant species are known. On the other hand, Cessator anachronicus, as well as the first reported fossil lycid larva herein described, can be readily assigned to an extant genus. The newly studied specimens have all the generic diagnostic characters of Cessator (Leptolycini) and are so similar to their extant congenerics that if individuals were not encased in amber, it would be impossible to determine that this species is extinct. The remarkable similarity of C. anachronicus and also C. brodzinskyi with recent congenerics is not exclusive to this genus and this pattern can be found in various other known beetles from Dominican amber. In fact, the taxonomic placement of Dominican amber inclusions in extant genera of plants and insects is a common aspect of this fossil deposit (see references further in the discussion). The other known paedomorphic Dominican amber Leptolycini-E. avus and an undescribed species of Leptolycini from Wu 44 -also have the same conspicuous similarity with their respective congenerics, possessing all the modern features present in extant species. Outside of the Leptolycini, only a few morphological differences can be seen in Plateros jardinesi Kazantsev, 2020 from the Early Miocene (23-15 Mya) Mexican Chiapas amber and its recent congeners, and also between the majority of lycid species from Baltic amber (ca. 30-50 Mya) and their recent congeners, suggesting morphological stasis in these groups too.
These observations conflict with the seemingly established notion that beetle lineages affected by the paedomorphic syndrome are hypothesized to speciate faster and diversify more rapidly than non-paedomorphic groups 26,[28][29][30]41 due to their low dispersal capacity because of morphological modifications attributed to the paedomorphic syndrome, including flightlesness in females 6 . Indeed, poorly dispersing beetle lineages have a greater tendency to diversify 41 . The reduced mobility, interrupted gene flow and consequently limited geographical range has been reported to increase allopatric speciation and endemism rates in paedomorphic lycids 8,28 . Studies on other animals have also associated higher speciation and diversification rates with increased morphological modifications and phenotypical diversifications 29,30,46,47 . It can be therefore anticipated that lineages affected by the paedomorphic syndrome are expected to present a more diverse and divergent morphology. However, the examples found in Cessator and other fossil Leptolycini seem to contradict these assumptions, since very little morphological variation and divergence was found in the reported groups.
How can we explain these controversial observations, at least for the Greater Antilles lycid genera from the Dominican amber? One possible explanation is the relatively young age within the range of uncertainty associated with the age estimates of the Dominican amber. A younger age of the fossil within an uncertainty interval simply reduces time available for the lineages to diversify and develop their morphological diversity. In the early phase of speciation, lineages may differ only genetically but remain morphologically similar 28 . This notion may be relevant in support for the minimal age estimate of the Dominican amber in the controversy that places its age somewhere between 13 and 30 Mya 31,48-55 , with even younger ages proposed by Braga et al. 56 . Many other Dominican amber insects, including beetles from various families, have also been described in extant genera and they also displayed little morphological variation from their modern relatives (see review in Hörnschemeyer et al. 57  Another explanation, or rather additional factor enforcing the first explanation, is a presumed microhabitat stability of Greater Antilles tropical forest leaf litter from Chattian in Oligocene (i.e., the oldest time estimate for Dominican amber) until present. Previous studies 28,57,61,62 suggested that microhabitat stability can be one of the factors that promote evolutionary stasis in beetles and other arthropods. The microhabitat conditions in the Greater Antilles, in which the Leptolycini can be found, with male adults flying in lower vegetation and larvae and females being found in leaf litter 40,63 , presumably persist since before the time of Dominican amber production. Dominican amber fossil plant records indicate stable composition of plant communities in the West Indies since the Miocene 64,65 , presumably harboring a similar leaf litter microhabitat structure where modern Leptolycini have been found.
While the interplay between the paedomorphosis and long-term persistence of microhabitats remains largely unknown and poorly discussed, the role of the presumed unchanged paleoecology of microhabitat could be more relevant than originally thought. The example of the Greater Antilles Cessator and the other known Leptolycini fossils may also indicate that groups affected by the paedomorphic syndrome, in such stable conditions, will not necessarily have a more divergent morphology linked to their presumed higher speciation and diversification rates. While there is some controversy as to whether or not higher diversification and speciation rates will necessary lead to morphological diversification (see discussions in Adams et al. 66 and Beltrán et al. 30 ), there are no studies similar to those cited above focused on relatively young group of paedomorphic insects, such as the Leptolycini.
It is important to stress that the scope of studies suggesting that paedomorphic Lycidae lineages do speciate rapidly has been limited, only focusing on groups that are entirely affected by paedomorphic processes, i.e., all ingroup taxa included in those studies were completely comprised of paedomorphic species 8,28 . In such studies, it is presumed that the paedomorphic syndrome evolved at the early stage of the evolutionary diversification in those groups, and all subsequent descendants (= terminals) simply retained the symplesiomorphic features associated with the syndrome. There are no studies on diversification and morphological variation for heterogenous paedomorphic groups (i.e., groups for which the paedomorphic syndrome has evolved independently more than once within the same lineage), such as presumably several South American paedomorphic Lycidae [18][19][20][21][22][23][24][25]67 . Further correlations between higher speciation and diversification rates and morphological diversity are still very limited in invertebrates, especially in beetles, and more in-depth and large-scale studies on these topics are yet to be proposed before broader extrapolations can be confidently made. www.nature.com/scientificreports/

Conclusion
The study of fossils is important not only because of the intrinsic improvement they generate on the taxonomic knowledge of a group, but also because they very often lead to clues to the evolutionary processes shaping the diversity of organisms. The description of Cessator anachronicus and of the first Cessator larva from Dominican amber represent further advances on the taxonomic knowledge of the poorly studied Leptolycini and in Lycidae overall, by providing further dating points that can be used to calibrate the phylogeny of the family. This study also provides further historical documentation of paedomorphosis in a Lycidae lineage from the West Indies, as well as evidence of morphological stasis in a group for which rapid acquisition of high phenotypic and morphological diversity was anticipated. Possible explanations for the observed morphological stasis in the Leptolycini involve the shorter period of time (ca. 15 myr) which elapsed from the time of Dominican amber deposition to present, i.e., not enough time to develop morphological diversity, and, more importantly, stability of the leaflitter and forest microhabitats for tens of million of years in the West Indies. Even though the paedomorphic syndrome and microhabitat stability are expected to be antagonistic evolutionary drivers given some already published evidence, in Cessator, their interaction resulted in a case of morphological stasis, in which extant and extinct species are nearly identical. While this study does not provide any definitive answer for the questions herein raised, we hope it will draw attention to the interplay of these evolutionary drivers and their effects in beetles' morphology evolution.

Material and methods
All four studied amber specimens are deposited in the Amber Collections of the American Museum of Natural History, New York, NY, USA (AMNH) and were sent in a loan for this study by Dr. David Grimaldi (curator). The exact locality of the specimens is unknown, and the only information provided regarding their age is that the samples are from the Oligocene-Miocene horizon. The specimens were studied under a LeicaWild M3C stereoscopic microscope with magnification up to 40× and identified using available literature 20,21,35,38,43,68 and by comparison of material with Leptolycini specimens assembled in the Montana Entomology Collection, Bozeman, MT, USA (MTEC). Adult morphological terminology follows Kazantsev 69 and Lawrence et al. 70 , and immature terminology follows Ferreira and Costa 71 .
For photographs, specimens were completely submerged in olive oil. Photos were taken using a Canon 6D DSLR using the lens MP-E 65 mm and a Stackshot-automated macro rail for focus stacking. Images were processed with Zerene Stacker software version 1.04. Enhancements to digital images were made in Adobe Photoshop CC 2021 and final plates prepared using Adobe Illustrator CC 2021.