Cuticular lipid diversification in Lasiommata megera and Lasiommata paramegaera: the influence of species, sex, and population (Lepidoptera: Nymphalidae)

Numerous studies have investigated the presence and the effectiveness of volatile pheromones in Lepidoptera. Conversely, very few studies have focused on the composition and the perception of the relatively low volatile components of cuticular mixtures. Yet, cuticular lipids are implied in the recognition processes of several solitary and social insects. In the present study, the cuticular signatures of the satyrid butterflies Lasiommata megera and Lasiommata paramegaera were examined by gas chromatography. General linear model and discriminant analyses on chemical data clearly revealed large differences between sexes, which showed the same diversification pattern in both species. Moreover, a strong diversification between the two species was found, as were differences among populations. These results represent a first step in demonstrating the communicative function of cuticular compounds in the L. megera / paramegaera complex. Moreover, the discrimination among different species and populations on the basis of cuticular mixtures could represent a platform for studying chemotaxonomy and chemical biogeography in butterflies, as already found in several other insect groups.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 703–710.     

Conservation biogeography of large Mediterranean islands. Butterfly impoverishment, conservation priorities and inferences for an ecologica "island paradigm"

Conservation biogeography is considered the Cinderella of biological conservation. Nevertheless biogeography provides the basis for establishing species distributions over space and time, therefore conservation priorities among areas and individual species. We demonstrate that there is no need to simplify analyses by using subsets of species (rare species, endemics) as surrogates. In doing so, we apply strict biogeographical techniques to determine butterfly impoverishment on three of the west Mediterranean's largest islands (Sardinia, Corsica and Sicily). The analyses performed on species, both collectively and individually, reveal that regional species richness in the Mediterranean zone can be largely predicted by latitude, altitude and latitudinal range (maximum minus minimum latitude), but that Sardinia and Corsica have clearly impoverished faunas. Logistic regression at individual species level demonstrates that several species, predicted to be present in these islands on the basis of their continental distributions, are actually absent. When compared with species that are present in these islands, such missing species are disclosed as having ecological traits which reduce their colonization capability. Probabilities of occurrence are calculated for each species on each island; they reflect the potential for each butterfly species to migrate to and colonise each island, and can be considered as a measure of conservation value. As such, species present on islands but having low immigration probabilities are predicted to represent isolated populations from the mainland that are unlikely to re-colonize the islands in the case of extinction. Island endemic species and races are shown to have lower occurrence probabilities compared to widespread species occurring on islands and illustrate the usefulness of occurrence probabilities for identifying isolated populations in need of conservation attention.     

Identifying zones of phenetic compression in West Mediterranean butterflies (Satyrinae): refugia,invasion and hybridization

Aim Distinct insular populations are generally considered important units for conservation. In island–mainland situations, unidirectional introgressive gene flow from the most abundant, typically continental, populations into the smaller island populations can erase native insular genetic units. As an indication of threat, the concept of phenetic slope is developed, a measure proportional to differentiation and to geographical proximity. Location The Western Mediterranean, including the following islands: Sardinia, Sicily, Corsica, Balearics, circum‐Italian, circum‐Sicilian and circum‐Sardo‐Corsican archipelagos. Eastern Europe is included for comparison. Methods Geometric morphometrics was applied to 2392 male genitalia of seven butterfly species groups. Geographic Information System techniques were used to depict the pattern in the distribution of morphotypes. The slope of variation in genital shape was computed to highlight geographical areas showing abrupt morphological changes. Correlation analyses were performed between the mean slope values across sea straits separating islands and nearest sources and ecological traits of the species that underlie their colonization and migration capacity. Results Phenetic slope analysis has revealed that the strait of Messina and the northern Tyrrhenian Sea support particularly contrasting populations. In these areas, mean slopes for species also correlated with certain ecological traits of the species. Sardinia emerges as the most stable refugium for ancestral mediterranean populations. Main conclusions There is strong support for the hypothesis that Italy has experienced invasion by populations from Eastern Europe with postglacial expansion of these populations across Italy. However, propagules are impeded from invading islands by the expanse of sea straits. Even so, sea straits are not invariably barriers. Our results suggest that wind direction in combination with habitat occupancy may have maintained ancestral insular populations in key locations distinguished by phenetic compression. We conclude that native insular populations acting as barriers to introgression in the areas showing particularly steep phenetic slopes deserve attention in conservation programmes.     

Similarities in the surnames of island and continental populations of the north-western Mediterranean area

This paper compares the structures of the surnames of 75 municipal populations living in six north-western Mediterranean regions. Its purpose is to unravel the relations between the local populations in Corsica and Sardinia and the links between these populations and those living in the Italian and French continental territory. On the basis of the matrix of similarity of surnames, some topological representations have been drafted showing the above-mentioned relations between populations under the light of their geographical position, their recent history and studies of genetic analysis. Corsica has an eterogeneous surname structure and evident similarity of the north with Tuscany and some centres of continental France. When only the populations of Sardinia were taken into consideration, it emerged that they differ among each other in relation to their geographical position and their history; when, instead, they were considered in relation to other populations outside the island, it was possible to observe that they form a highly different cluster. This study also identified many differences in the analysed geographical areas of Sardinia. In the minor islands - Elba, Giglio, Capraia - the structure of the surnames has a Tuscan origin as well as some similarity with other geographically distant areas, as in the case of the island of Giglio, if compared with some communities of Liguria.     

Effects of large-scale cattle grazing on populations of Coenonympha glycerion and Lasiommata megera (Lepidoptera:Satyridae)

The effects of large-scale cattle grazing by herdsmen (Allmende) on the populations of two butterfly species, Coenonympha glycerion and Lasiommata megera, were analysed on a study plot in the foothills of the Eastern Carpathians, Republic of Ukraine, in the summer and autumn of 1995. In all, 280 individuals of C. glycerion (115 females) and 230 individuals of L. megera (61 females) were individually marked. At recapture rates of 35 to 39%, maximum population size was calculated at 410 and 434 individuals. C. glycerion reacted to high densities by shifting to smaller neighbouring habitats. An increased emigration rate was recorded for L. megera when the population maximum was reached.Both species essentially require habitats formed by the activity of cattle. C. glycerion significantly preferred south facing woodland margins, close to moderately and infrequently grazed grassland. Heavily grazed areas were avoided. L. megera mainly occupied well-trod cattle paths or landslips with rather sparse vegetation, induced by cattle. Preferred habitats are characterized by linear structures for patrolling, steep slope areas and a wide range of nectar resources. Smaller, sporadically used cattle paths did not show these features and were occupied mainly by males at times of high population densities. Both species depend on patchy habitat mosaics which will be lost by intensification as well as by a complete cessation of grazing. The low-intensity, large-scale system utilized in the Eastern Carpathians is therefore favourable to both species.     

The biogeography of non-marine molluscs in the Tuscan Archipelago reveals combined effects of current eco-geographical drivers and paleogeography

We investigated the role of present (Recent) and historical (Pleistocene, i.e., Würmian Last Glacial Maximum) eco-geographical variables on the richness and diversity of non-marine molluscs in the Tuscan Archipelago, as well as inter-island faunal dissimilarity and relationships with source pools (Sardinia and Corsica, Tuscany). The association between species richness and present and historical eco-geographical variables were assessed with Spearman’s rank correlation test, while faunal dissimilarity both between islands and with their source pools was analyzed through beta-diversity partitioning (Sørensen index and its nestedness and turnover component) with UPGMA clustering tested with a multiscale bootstrap procedure. Non-metric multidimensional scaling in RGB color space was also used. Multiple regressions on distance matrices were then applied to explain assemblage composition between islands. Analyses were performed on all species and on all species except aliens. The overall framework showed the combined effects of current eco-geographical and paleogeographical imprints on non-marine malacofauna in the Tuscan Archipelago. However, excluding aliens, differences in species spatial turnover showed a clear correlation with Pleistocene inter-island distances, evidence of stronger historical biogeographical relationships between islands. This may indicate that widespread native species established their distribution during the Pleistocene, while alien species spread into the Tuscan Archipelago through stochastic and human-mediated dispersion events in recent times. Interestingly, Giglio’s relationships do not agree with the most accepted paleogeographical model, suggesting that this island might have been connected to the Tuscan mainland during the Würmian Last Glacial Maximum. An in-depth revision of the paleogeographic framework of the northern Tyrrhenian is therefore called for.     

Volatile chemical composition does not support a native status of the cryptogenic Bupleurum fruticosum (Apiaceae) in peninsular Italy

Cryptogenic (or doubtfully native) species could have relevant implications in biogeography and in nature conservation. Bupleurum fruticosum is native in Sicily, Sardinia, and Liguria, whereas the single Tuscan population is considered cryptogenic. Given its pharmacological activity, several studies investigated the essential oil composition of this species, but an analysis of geographic pattern of the chemical composition was never performed. In the present study, the essential oil composition of the Tuscan population was investigated for the first time, and a comparison with other populations from literature was performed in order to provide useful data to disentangle its cryptogenic status. Our results highlight a major similarity of plants from Tuscany with geographically distant populations from Portugal and France, whereas other Italian populations show a very different chemical composition. These results do not support a native status of Tuscan population, suggesting an ancient human introduction. Our analysis also revealed a high percentage of (Z)-β-ocimene in stems of plants from Tuscany.     

The role of the North Atlantic Oscillation in controlling U.K. butterfly population size and phenology

1. The North Atlantic Oscillation (NAO) exerts considerable control on U.K. weather. This study investigates the impact of the NAO on butterfly abundance and phenology using 34 years of data from the U.K. Butterfly Monitoring Scheme (UKBMS).2. The study uses a multi-species indicator to show that the NAO does not affect overall U.K. butterfly population size. However, the abundance of bivoltine butterfly species, which have longer flight seasons, were found to be more likely to respond positively to the NAO compared with univoltine species, which show little or a negative response.3. A positive winter NAO index is associated with warmer weather and earlier flight dates for Anthocharis cardamines (Lepidoptera: Pieridae), Melanargia galathea (Lepidoptera: Nymphalidae), Aphantopus hyperantus (Lepidoptera: Nymphalidae), Pyronia tithonus (Lepidoptera: Nymphalidae), Lasiommata megera (Lepidoptera: Nymphalidae) and Polyommatus icarus (Lepidoptera: Lycaenidae). In bivoltine species, the NAO affects the phenology of the first generation, the timing of which indirectly controls the timing of the second generation.4. The NAO influences the timing of U.K. butterfly flight seasons more strongly than it influences population size.     

Comparative phenology of four satyrine butterflies inhabiting dry grasslands in Sweden

The phenology of the satyrine butterflies Coenonympha pamphilus L., Lasiommata megera L., Hipparchia semele L. and Maniola jurtina L. was compared under seminatural conditions with Festuca ovina as the food-plant. Coenonympha pamphilus shows striking phenotypic plasticity as revealed by initiating cohorts at successive dates during the whole flight period. Growth rate is finely tuned depending on season, and larvae can hibernate in any of the three first instars, but also aestivate when 'surplus'time is available. Hipparchia semele and M. jurtina have reduced growth rates as last instar larvae at the beginning of summer. Lasiommata megera , which has an obligate bivoltine life cycle, shows the highest overall growth rate of all four species. Results suggest that egg size variation has consequences for the life cycles of these species.     

Variation in the genitalia of the butterfly Maniola jurtina (Lepidoptera: Satyrinae)

The genilalia of animals, particularly insects, are extremely useful taxonomic characters, for they are highly conserved within species yet generally diverge rapidly during specialion: often the only reliable means of separating sibling species is via the morphology of the genilalia. A number of explanations have been proposed to explain this pattern of variation, the most prominent of which is the 'lock-and-key' hypothesis. This hypothesis and others are tested using the meadow brown butterfly, Maniola jurtina (L.), which exhibits variation in the male genitalia. A novel technique is described which enables dissection of copulating couples. Sampling and dissection of male butterflies from 14 populations in southern England was carried out to quantify variation in the genitalia. Mating success and the strength of the male-female bond during copulation was assessed in relation to the dimensions of the male genitalia.
The most variable portions of the male genitalia are the distal and dorsal margins of the paired valves. Contrary to the lock-and-key hypothesis, and the more recent sexual selection model, evidence is presented that the most variable portions of the genitalia have no apparent function during copulation, and therefore may not be subject to direct selection. I suggest that neutrality to selection was essential for the evolution of the wide variety in genital morphology currently found in the Lepidoptera.     

Satyrinae butterflies from Sardinia and Corsica show a kaleidoscopic intraspecific biogeography (Lepidoptera,Nymphlidae)

The Mediterranean islands of Sardinia and Corsica are known for their multitude of endemics. Butterflies in particular have received much attention. However, no comprehensive studies aiming to compare populations of butterflies from Sardinia and Corsica with those from the neighbouring mainland and Sicily have been carried out. In the present study, the eleven Satyrinae species inhabiting Sardinia and Corsica islands were examined and compared with continental and Sicilian populations by means of geometric morphometrics of male genitalia. Relative warp computation, discriminant analyses, hierarchical clustering, and cross‐validation tests were used to identify coherent distributional patterns including both islands and mainland populations. The eleven species showed multifaceted distributional patterns, although three main conclusions can be drawn: (1) populations from North Africa and Spain are generally different from those belonging to the Italian Peninsula; (2) populations from Sardinia and Sicily often resemble the North Africa/Spain ones; Corsica shows transitional populations similar to those from France; and (3) sea barriers represent filters to dispersal, although their efficacy appears to be unrelated to their extension. Indeed, the short sea straits between Sardinia and Corsica and between Sicily and the Italian Peninsula revealed a strong effectiveness with respect to preventing faunal exchanges; populations giving onto sea channels between Corsica and Northern Italy and between Sicily and Tunisia showed a higher similarity. A comparison of island and mainland distributions of the eleven taxa have helped to unravel the complex co‐occurrence of historical factors, refugial dynamics, and recent (post‐glacial) dispersal with respect to shaping the populations of Mediterranean island butterflies. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 195–212.     

Phylogenetic island disequilibrium: evidence for ongoing long‐term population dynamics in two Mediterranean butterflies

Aim Our aims were to verify the existence of phylogenetic disequilibrium between butterfly lineages at the subcontinental scale for islands and the nearest mainland and to test the capacity of islands for hosting ancestral populations of butterflies and the significance of such relict populations. Location The western Mediterranean continental area of Europe and North Africa together with several large and small islands (Balearics, Tuscan Archipelago, Aeolian Archipelago, Capri, Sardinia, Sicily, Corsica). Methods Using geometric morphometrics, the shape of male genitalia was analysed in two common butterflies (Pyronia cecilia and Pyronia tithonus), whose spatial heterogeneity in the Mediterranean region has recently been described. Observed patterns in genital shapes were compared with shapes predicted for islands and fossil islands to assess the contribution of historical and current events in accounting for the transition from a refugial model to an equilibrium model. Measurements were taken for 473 specimens in 90 insular and mainland sites. Results The shape of the genitalia of populations of most islands differed substantially from that predicted by the equilibrium hypothesis while closely fitting the refugial hypothesis. The comparison between different models strongly suggests that islands maintain ancestral lineages similar to those living in Spain (P. cecilia) and France (P. tithonus). A high correlation between observed and predicted patterns on islands and fossil islands occurs during the first steps of modelled introgressive hybridization while the following steps exposed a successively lower fit, suggesting that the process from a refugial to an equilibrium situation is highly skewed towards an earlier non‐equilibrium. Main conclusions The observed non‐equilibrium pattern supports the refugial hypothesis, suggesting that an ancestral lineage was originally distributed from Spain to Italy, and also occupied offshore islands. This lineage, replaced in Italy, has persisted on the islands owing to their isolation. A comparison of the distribution patterns for genetic and morphometric markers in several species indicates that the situation highlighted for Pyronia may represent a common biogeographic feature for many Mediterranean butterflies.     

Island size is not the only consideration. Ranking priorities for the conservation of butterflies on Italian offshore islands

Ecological and historical factors virtually create a unique faunal assemblage on each island. From this perspective every island deserves protection. However, economic limitations usually restrict conservation efforts to particularly important areas. As part of the SLOSS issue (the relative importance of single large or several small areas), there is the long debated question of whether it is better to protect few large areas (islands) or several small areas (islands). Here, we assess the butterfly faunas of the Italian offshore islands, using several biodiversity measures, in order to highlight priorities for conserving butterfly richness, rarity and endemicity. First, the nested pattern of butterfly fauna was investigated to determine the relative importance of large and small islands. Then, residuals were assessed for the species-area relationship and for multiple regressions of richness, rarity and endemicity against geographic variables. Subsequently, two other indices were calculated: Biodiversity Conservation Concern and an index scoring islands in the order that maximizes the cumulative percentage of total, endemic, and rare species. The results clearly indicate that although greatest concern is for the island having the largest butterfly fauna in the sample (Elba), the importance of several small islands should not be ignored. This is primarily due to the substantial impact of source areas and consequently the occurrence of several rare and endemic species occurring on small islands as well as on large islands.     

Dynamics of mate-searching behaviour in a hilltopping butterfly, Lasiommata megera (L.): the effects of weather and male density

By studying a hilltopping population of the satyrine Lasiommata megera in southern Sweden, the effects of density and meteorological conditions on mate-searching behaviour were investigated. Lasiommata megera males switched between stationary and vagrant behaviour, and the behaviour adopted was correlated to meteorological conditions; more males were stationary at low temperatures, low irradiation levels and high wind speeds than vice versa. Body temperature measurements indicated that these factors were important in deciding the body temperature and thereby the flight activity of males. Male density did not significantly influence mate-locating behaviour. The tendency of males to hilltop and their distribution around the hill were strongly influenced by weather. The hilltopping behaviour of this species did not adhere to an all-or-none pattern, but instead males, as well as the stations they used, were found at increasingly higher elevations on the hill with increasing temperatures and decreasing wind speeds. Moreover, males tended to use the lee side and sun-exposed side of the hill. Females, of which the majority were already mated, also showed this distribution around the hill, but preferred lower elevations than males. Release experiments did not reveal any propensity of mated females to fly uphill. Such a propensity was shown by released virgin females.     

An inflated conservation load for European butterflies: increases in rarity and endemism accompany increases in species richness

The addition of species to the European butterfly list since 1983 has resulted in a number of highly significant changes. Most important are the increases in the number and proportion of endemics and of rare species, and a regional excess of species and endemics for southern Europe compared to northern Europe. There is also a surplus of Lycaenidae and Satyridae compared to other families, and an increase in species per genus associated with the reduction in genera. These additions raise two issues. First, the potential conservation load for European butterflies is inflated at species level. This is especially the case for southern Europe, which has disproportionate increases in rare and endemic species, more particularly if rarity and endemism are found to equate with threat of extinction. Second, the inflation in rarity and endemism suggests that there is a trend to promote ever more local populations (races, subspecies) to species. The taxonomic status of species being added to the list, a quarter of which are regarded as doubtful, is increasingly difficult to determine. Consequently, there is a danger that this may call into question the validity and objectivity of taxonomic practices, and of databases dependent on them, used by conservation. Revision of higher and lower butterfly taxa is urgently required.     

Taxonomic Boundaries and Craniometric Variation in the Treeshrews (Scandentia,Tupaiidae) from the Palawan Faunal Region

The taxonomy of treeshrews (Order Scandentia) has long been complicated by ambiguous morphological species boundaries, and the treeshrews of the Palawan faunal region of the Philippines are no exception. Four named forms in the genus Tupaia Raffles, 1821, have been described from four island groups based on subtle qualitative morphological characters, and as many as three distinct species have been recognized. A recent molecular phylogenetic study of relationships among Tupaia species suggests that the two currently-recognized treeshrew species from the Palawan faunal region diverged very recently relative to other sister-species divergences within the genus and may not represent species-level taxonomic entities. Here we review the taxonomic and biogeographic histories of the Tupaia taxa from this region. We also collected craniodental data from 133 skulls of all four named forms, representing five island populations, and conducted univariate and multivariate analyses on these data. Our morphometric results are consistent with molecular results, further suggesting that there is insufficient evidence to recognize T. moellendorffi Matschie, 1898, as a separate species from T. palawanensis Thomas, 1894. Our analyses also revealed a craniodentally divergent population from the island of Balabac, which has never been considered a distinct subspecies (or species) from the population on Palawan. These results have conservation implications for the island populations in our analyses, but additional surveys and molecular evidence will be required to fully assess conservation priorities for the treeshrews of the Palawan faunal region.     

A geographic mosaic of evolutionary lineages within the insular endemic newt Euproctus montanus

Islands are hotspots of biodiversity, with a disproportionately high fraction of endemic lineages, often of ancient origin. Nevertheless, intra‐island phylogeographies are surprisingly scarce, leading to a scanty knowledge about the microevolutionary processes induced on island populations by Plio‐Pleistocene climatic oscillations, and the manner in which these processes contributed to shape their current genetic diversity. We investigated the phylogeography, historical demography and species distribution models of the Corsican endemic newt Euproctus montanus (north‐western Mediterranean). As for many island endemics, the continuous distribution of E. montanus throughout its range has hitherto been considered as evidence for a single large population, a belief that also guided the species' categorization for conservation purposes. Instead, we found a geographic mosaic of ancient evolutionary lineages, with five main clades of likely Pliocene origin (2.6–5.8 My), all but one restricted to northern Corsica. Moreover, the copresence between main lineages in the same population was limited to a single case. As also suggested by growing literature on intra‐island phylogeographic variation, it seems that the extensive use of simplifying assumption on the population structure and historical demography of island populations—both in theoretical and applicative studies—should be carefully reconsidered, a claim that is well exemplified by the case presented here.     

The human settlement of Corsica: Palaeogeographic and tectonic considerations

There are no marks of human settlement in Corsica during the Paleolithic age. This paper is a reflection on the following question: could Man have come to this island before the Neolithic age? The possibilities of the existence of an emerged region between Corsica and the mainland are considered.The Quaternary fauna suggests that an isthmus was possible during the Early Würm, unless the animals in the würmian sediments had come into the island previously.The bathymetry around Corsica shows that the shallowest region is between Corsica and Tuscany, via Capraja, Elba, Pianosa islands. But this sea bottom is deeper than the lower levels known or estimated for glacio-eustatical regressions during the Quaternary era. Thus, emersion of this region should have required tectonical movements. The neotectonic is known in Corsica and in perityrrhenian regions; the present bathymetry of the area which may have been emerged should be explained by movements of a credible rate.     

Using genetics to inform re-introduction strategies for the Chequered Skipper butterfly (Carterocephalus palaemon, Pallas) in England

The Chequered Skipper butterfly (Carterocephalus palaemon) is extinct in England but extant in Scotland and Continental Europe. The possibility of re-introducing the species is under active consideration by conservation bodies, but ecological differences between Scottish and English populations raise the question of which populations should donate individuals, Continental European, or Scottish? We used mitochondrial DNA sequences (CO I, CO II and Cyt b) from potential donor populations to test the hypothesis that ecological differences could have arisen as a result of differing routes of post-glacial colonisation from separate refugia and subsequent isolation of UK populations. Shared haplotypes between populations in Belgium, Norway, Scotland and England provides no evidence to support the hypothesis that populations in Scotland result from an alternative post-glacial colonisation route. As the genetic evidence remains equivocal we suggest that choice of donor stock for a re-introduction to England should be made primarily on ecological grounds.     

Nestedness of butterfly assemblages in the Zhoushan Archipelago,China: area effects,life-history traits and conservation implications

The nested subset pattern (nestedness) of faunal assemblages has been a research focus in the fields of island biogeography and conservation biology in recent decades. However, relatively few studies have described nestedness in butterfly assemblages in oceanic archipelago systems. Moreover, previous studies often quantified nestedness using inappropriate nestedness metrics and random fill algorithms with high Type I errors. The aims of this study are to examine the existence of nestedness and underlying causal mechanisms of butterfly assemblages in the Zhoushan Archipelago, China. We used the line-transect method to determine butterfly occupancy and abundance on 42 study islands from July to August 2014. We obtained butterfly life-history traits (wingspan, body weight and minimum area requirement) by field work and island geographical features (area and isolation) from the literature. We used the recently developed metric WNODF to estimate nestedness. Partial Spearman rank correlation was used to evaluate the associations of nestedness and island geographical features as well as butterfly life-history traits related to species extinction risk and colonization ability. The butterfly assemblages were significantly nested. Island area and minimum area requirement of butterflies were significantly correlated with nestedness after controlling for other independent variables. In contrast, the nestedness of butterflies did not appear to result from passive sampling or selective colonization. However, multi-year studies are needed to confirm that target effects are not muddling these results. Our results indicate that selective extinction may be the main driver of nestedness of butterfly assemblages in our study system. From a conservation viewpoint, we should protect both large islands and species with large area requirement to maximize the number of species preserved.