Distribution and species richness of woody dryland legumes in Baja California,Mexico

Abstract. We analysed the biogeographic patterns of woody legumes in the Baja California peninsula, NW Mexico. From the specimen labels of eight herbaria, we digitized 4205 records from 78 species, and projected them onto a grid of 205 cartographic cells (20’ longitude × 15’ latitude). Most species followed distribution patterns that coincide with floristic subdivisions of the peninsula. Endemism is high, reaching 60–70% in the centre of the peninsula, where the driest deserts are found and where significant floristic changes took place during Pleistocene glacial events. The number of cartographic cells (i.e. their geographic ranges) were log‐normally distributed, as has been reported for many other taxa. Floristic richness was found to be clumped around some cells where the observed richness is significantly higher than could be expected from chance variation. We tested the hypothesis that these ‘hotspots’ could be attributable to great collection efforts or to large land surfaces, but we still found 16 cells where richness is significantly high once these two factors are accounted for. Species richness and micro‐endemism increase towards the south, conforming to Rapoport's rule that predicts that species ranges become smaller towards the equator while richness increases. The floristic hotspots for woody legumes in Baja California occur in the Cape Region and along the Sierra de la Giganta in the southern Gulf Coast, where 77% of the total peninsular legume flora can be found. These hotspots are mostly unprotected, and should be considered priority areas for future conservation efforts.     

New records of large branchiopods (Branchiopoda: Anostraca,Notostraca, and Spinicaudata) in Mexico

This paper reports new distribution records of large branchiopods for Mexico following a three year survey of the Baja California peninsula. The occurance of the anostracans Thamnocephalus mexicanus (Linder, 1941) and T. platyurus (Packard, 1877), the notostracan Lepidurus lemmoni (Holmes, 1894), and the spinicaudatans Eulimnadia cylindrova (Belk, 1989) and E. texana (Packard,1871), all represent the first records for the peninsula. An undescribed species of the anostracan genus Streptocephalus is recorded from the state of Baja California (Norte). The occurrence of the notostracan genus Triops and four other anostracan species on the peninsula is also confirmed. The conchostracan Cyclestheria hislopi (Baird, 1859) is recorded from the state of Quintana Roo. The collections of Lepidurus and Cyclestheria are the first records for México. These records increase the number of species of large branchiopods reported fromMéxico to 36: 20 Anostraca, 3 Notostraca, 11 Spinicaudata, and 2Laevicaudata. This revised version was published online in August 2006 with corrections to the Cover Date.     

Island biogeography of bats in Baja California, Mexico: patterns of bat species richness in a near-shore archipelago

Aim We studied the relationship between the size and isolation of islands and bat species richness in a near‐shore archipelago to determine whether communities of vagile mammals conform to predictions of island biogeography theory. We compared patterns of species richness in two subarchipelagos to determine whether area per se or differences in habitat diversity explain variations in bat species richness. Location Islands in the Gulf of California and adjacent coastal habitats on the Baja California peninsula in northwest Mexico. Methods Presence–absence surveys for bats were conducted on 32 islands in the Gulf of California using acoustic and mist‐net surveys. We sampled for bats in coastal habitats of four regions of the Baja peninsula to characterize the source pool of potential colonizing species. We fitted a semi‐log model of species richness and multiple linear regression and used Akaike information criterion model selection to assess the possible influence of log₁₀ area, isolation, and island group (two subarchipelagos) on the species richness of bats. We compared the species richness of bats on islands with greater vegetation densities in the southern gulf (n = 20) with that on drier islands with less vegetation in the northern gulf (n = 12) to investigate the relationship between habitat diversity and the species richness of bats. Results Twelve species of bats were detected on islands in the Gulf of California, and 15 species were detected in coastal habitats on the Baja peninsula. Bat species richness was related to both area and isolation of islands, and was higher in the southern subarchipelago, which has denser vegetation. Log₁₀ area was positively related to bat species richness, which increased by one species for every 5.4‐fold increase in island area. On average, richness declined by one species per 6.25 km increase in isolation from the Baja peninsula. Main conclusions Our results demonstrate that patterns of bat species richness in a near‐shore archipelago are consistent with patterns predicted by the equilibrium theory of island biogeography. Despite their vagility, bats may be more sensitive to moderate levels of isolation than previously expected in near‐shore archipelagos. Differences in vegetation and habitat xericity appear to be associated with richness of bat communities in this desert ecosystem. Although observed patterns of species richness were consistent with those predicted by the equilibrium theory, similar relationships between species richness and size and isolation of islands may arise from patch‐use decision making by individuals (optimal foraging strategies).     

Endemic regions of the vascular flora of the peninsula of Baja California,Mexico

Question: Can we recognize areas of high endemism and high endemic richness, using data from collections, and what are the ecological variables that best explain these areas? Location: Peninsula of Baja California, Mexico. Methods: We analysed the distribution of 723 endemic vascular plants species along the peninsula of Baja California and neighbouring islands distributed in 218 cartographic cells 15’ x 20’ in size. By means of a residual analysis, we identified areas of significantly high endemic species richness, and we calculated the degree of endemicity (or rarity) in each cell by giving to each species a weight factor inversely proportional to the land area it covers. Results: Nine regions of high‐endemicity and/or high endemic species richness were found. Discussion and conclusions: The analyses of rarity and endemic species richness showed two contrasting scenarios: High endemicity values in oceanic and sky islands accounts for a high number of species with a restricted distribution, promoted most likely by genetic isolation and high environmental heterogeneity. High endemic richness along the peninsular coast is related to ecotonal transition along vegetation types. After correcting for collection effort (i.e. the number of specimens collected within a cell), we found the phytogeographic region and altitudinal heterogeneity to be the variables that best predicted endemic richness. Both high endemism and high endemic richness have distinct geographic patterns within our study region. The nine endemic regions provide elements for priority definitions in future conservation programs.     

Does versatility as measured by geographic range,bathymetric range and morphological variability contribute to taxon longevity?

Aim This paper aims to examine the relationship between versatility as measured by geographic range, bathymetric range and morphological variability (species and subspecies richness and the occurrence of morphologically highly variable populations), and the geologic longevities of trachyleberidid ostracode species and genera, while accounting for sampling biases and other confounding factors. Location Global. Methods A large database of occurrence records of species of the family Trachyleberididae s.l. was analysed. The relationships between genus and species longevity and the above mentioned variables were examined singly and in concert. Re‐analyses of subsets of data and rarefaction techniques were employed to account for sampling biases, while randomization was used to account for autocorrelation of variables. Results The mean number of occurrence records, and latitudinal and longitudinal ranges, were strongly and positively correlated with genus and species longevities. The number of bathymetric zones occupied by genera had no consistent bearing on their longevities, but species data subsets tended to indicate significant positive relationships between bathymetric range and longevities. Species richness was significantly and positively correlated with genus longevities. Species and genera with subspecies and species with high morphological variability all had significantly greater longevities. Genus‐level characteristics can be explained largely by species‐level characteristics, including longevity, latitudinal ranges and bathymetric ranges to a lesser degree. However, genus longevity was best explained by species richness and genus age, even for extinct genera, while species longevity was best explained by species latitudinal range. Main conclusions In spite of the incompleteness of the fossil record, we can control for biasing factors and still confidently draw the conclusion that both ecological and evolutionary versatility contribute to lineage longevity, beyond the shorter temporal observation windows available to most ecological studies.     

The peninsula effect on species diversity: a reassessment of the avifauna of Baja California

The peninsula effect, a decrease in species diversity from the base to the tip of peninsulas, has been proposed to explain the relatively poor species diversity of mammals on North American peninsulas. Subsequent work has questioned both the existence of peninsular declines in diversity, as well as the proposed cause (immigration-extinction dynamics). Previous studies of the Baja California avifauna have shown a gradual decrease in the diversity of breeding birds from the base to the tip of the peninsula. Using newly published data on the breeding land birds, I found a decrease only from the base to the middle of the peninsula, with a slight increase in diversity from the middle to the tip. This result is similar to that for other highly vagile taxa (e.g., Chiroptera. Lepidoptera) and is largely due to the coneave diversity gradient of montane species along the peninsula. Habitat associations of the Baja avifauna and the location of potential source populations suggest that: 1) local habitat heterogeneity is likely the single most important factor influencing the avian diversity gradient along the peninsula; and 2) limited immigration of Neotropical species from mainland areas, and of Nearctic species from the base of the peninsula to the montane southern tip is partly responsible for the form of the diversity gradient along the southern half of the peninsula. My results along with those from previous studies, suggest that rather than colonization/extinction dynamics, habitat heterogeneity and the vagility of the taxa considered have the greatest impact on the observed patterns of species diversity along peninsulas,     

Late Pleistocene to Holocene distributional stasis in scorpions along the Baja California peninsula

The biota of the Baja California peninsula (BCP) assembled in response to a complex history of Neogene tectonics and Quaternary climates. We constructed species distribution models (SDMs) for 13 scorpion species from the BCP to compare current suitable habitat with that at the latest glacial maximum about 21 000 years ago. Using these SDMs, we modelled climatic suitability in relation to latitude along the BCP. Our SDMs suggested that most BCP scorpion distributions have remained remarkably conserved across the latest glacial to interglacial climatic transformation. Three areas of climatic suitability coincide remarkably well with genetic discontinuities in other co‐distributed taxa along the BCP, indicating that long‐term persistence of zones of abrupt climatic transition offer a viable alternative, or synergistic enhancement, to hypotheses of trans‐peninsular seaways as drivers of peninsular divergences. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 450–461.     

Genetic differentiation and demographic history in Drosophila pachea from the Sonoran Desert

Genetic variation at six microsatellite DNA loci and a segment of the mitochondrial cytochrome oxidase subunit I (COI) locus was used to estimate gene flow, population structure, and demographic history in the cactophilic Drosophila pachea from the Sonoran Desert of North America, a species that shows a strict association with its senita host cactus (genus Lophocereus). For microsatellite analyses, thirteen populations of D. pachea were sampled, five in mainland Mexico and the southwestern USA, and eight on the Baja California (Baja) peninsula, covering essentially the entire range of the species. Analysis of molecular variance (AMOVA) of microsatellite data revealed that populations from both the mainland and the Baja peninsula generally showed little structure, although there were a few exceptions, suggesting some local differentiation and restriction of gene flow within both regions. Pairwise comparisons of F(ST) among each of the mainland and Baja populations showed evidence of both panmixia and population subdivision. AMOVA performed on grouped populations from both the mainland and Baja, however, revealed significant partitioning of genetic variation among the two regions, but no partitioning among localities within each region. Bayesian skyline analyses of the COI data set, consisting of four mainland and seven peninsular populations, revealed population expansions dating to the Pleistocene or late Pliocene in D. pachea from both regions, although regional differences were seen in the estimated timing of the expansions and in changes in effective population size over time.     

A checklist of helminth parasites of Elasmobranchii in Mexico

A comprehensive and updated summary of the literature and unpublished records contained in scientific collections on the helminth parasites of the elasmobranchs from Mexico is herein presented for the first time. At present, the helminth fauna associated with Elasmobranchii recorded in Mexico is composed of 132 (110 named species and 22 not assigned to species), which belong to 70 genera included in 27 families (plus 4 incertae sedis families of cestodes). These data represent 7.2% of the worldwide species richness. Platyhelminthes is the most widely represented, with 128 taxa: 94 of cestodes, 22 of monogeneans and 12 of trematodes; Nematoda and Annelida: Hirudinea are represented by only 2 taxa each. These records come from 54 localities, pertaining to 15 states; Baja California Sur (17 sampled localities) and Baja California (10), are the states with the highest species richness: 72 and 54 species, respectively. Up to now, 48 elasmobranch species have been recorded as hosts of helminths in Mexico; so, approximately 82% of sharks and 67% of rays distributed in Mexican waters lack helminthological studies. The present list provides the host, distribution (with geographical coordinates), site of infection, accession number in scientific collections, and references for the parasites. A host-parasite list is also provided.     

SELECTION FOR SEXUAL ISOLATION BETWEEN GEOGRAPHIC FORMS OF DROSOPHILA MOJAVENSIS. I. INTERACTIONS BETWEEN THE SELECTED FORMS

Drosophila mojavensis comprises three geographic forms occurring in the United States, the Baja California peninsula, and mainland Mexico. Peninsular and mainland forms were selected for increased sexual isolation from each other, while controls were maintained with maximum outbreeding. Response to selection was highly asymmetrical in that isolation was very high between selected peninsular males and mainland females, but nonexistent between selected mainland males and peninsular females. The heightened isolation is primarily due to some change in the peninsular males.     

High Antipredatory Efficiency of Insular Lizards: A Warning Signal of Excessive Specimen Collection?

We live-captured lizards on islands in the Gulf of California and the Baja California peninsula mainland, and compared their ability to escape predation. Contrary to expectations, endemic lizard species from uninhabited islands fled from humans earlier and more efficiently than those from peninsular mainland areas. In fact, 58.2% (n=146) of the lizards we tried to capture on the various islands escaped successfully, while this percentage was only 14.4% (n=160) on the peninsular mainland. Separate evidence (e.g., proportion of regenerated tails, low human population at the collection areas, etc.) challenges several potential explanations for the higher antipredatory efficiency of insular lizards (e.g., more predation pressure on islands, habituation to humans on the peninsula, etc.). Instead, we suggest that the ability of insular lizards to avoid predators may be related to harvesting by humans, perhaps due to the value of endemic species as rare taxonomic entities. If this hypothesis is correct, predation-related behavioral changes in rare species could provide early warning signals of their over-exploitation, thus encouraging the adoption of conservation measures.     

Structure of Benthic Diatom Assemblages from a Mangrove Environment in a Mexican Subtropical Lagoon1

The taxonomic list and the structure of benthic diatom assemblages occurring in fine sediments (silt and sand) from the mangrove forest of the Balandra lagoon in Baja California Sur, Mexico was determined based on seasonal samplings for one year. Assemblage structure was analyzed using several ecological indices for estimating diversity (H'), dominance (REDI), equitability, and similarity. A total of 230 diatom taxa were identified and include 109 new records for the Baja California peninsula coast. Taxa representative of highly productive and hypersaline environments were common. Assemblages were characterized by a few abundant species and many uncommon or rare taxa. High diatom diversity estimates at all sampling sites during all seasons suggest that diatom assemblages in sediments of the Balandra lagoon represent a quasi-pristine environment.     

Historical and recent processes shaping the geographic range of a rocky intertidal gastropod: phylogeography,ecology, and habitat availability

Factors shaping the geographic range of a species can be identified when phylogeographic patterns are combined with data on contemporary and historical geographic distribution, range‐wide abundance, habitat/food availability, and through comparisons with codistributed taxa. Here, we evaluate range dynamism and phylogeography of the rocky intertidal gastropod Mexacanthina lugubris lugubris across its geographic range – the Pacific coast of the Baja peninsula and southern California. We sequenced mitochondrial DNA (CO1) from ten populations and compliment these data with museum records, habitat availability and range‐wide field surveys of the distribution and abundance of M. l. lugubris and its primary prey (the barnacle Chthamalus fissus). The geographic range of M. l. lugubris can be characterized by three different events in its history: an old sundering in the mid‐peninsular region of Baja (~ 417,000 years ago) and more recent northern range expansion and southern range contraction. The mid‐peninsular break is shared with many terrestrial and marine species, although M. l. lugubris represents the first mollusc to show it. This common break is often attributed to a hypothesized ancient seaway bisecting the peninsula, but for M. l. lugubris it may result from large habitat gaps in the southern clade. Northern clade populations, particularly near the historical northern limit (prior to the 1970s), have high local abundances and reside in a region with plentiful food and habitat – which makes its northern range conducive to expansion. The observed southern range contraction may result from the opposite scenario, with little food or habitat nearby. Our study highlights the importance of taking an integrative approach to understanding the processes that shape the geographic range of a species via combining range‐wide phylogeography data with temporal geographic distributions and spatial patterns of habitat/food availability.     

Studies on California ants: a review of the genus Temnothorax (Hymenoptera,Formicidae)

The following ten new species of the ant genus Temnothorax are described and illustrated: T. anaphalantus (California, Baja California), T. arboreus (California), T. caguatan (Oregon, California, Baja California), T. morongo (California, Baja California), T. myrmiciformis (California, Baja California), T. nuwuvi (Nevada), T. paiute (California, Nevada), T. pseudandrei (Arizona, California), T. quasimodo (California) and T. wardi (California). A key to workers of the twenty-two Temnothorax species known or expected to occur in California is provided.     

Ants and people: a test of two mechanisms potentially responsible for the large‐scale human population–biodiversity correlation for Formicidae in Europe

Aim: Recent coarse‐scale studies have shown positive relationships between the biodiversity of plants/vertebrates and the human population. Little is known about the generality of the pattern for invertebrates. Moreover, biodiversity and human population might correlate because they both covary with other factors such as energy availability and habitat heterogeneity. Here we test these two non‐mutually exclusive mechanisms with ant species‐richness data from the Fauna Europaea. Location Forty‐three European countries/regions. Methods We derived mixed models of total, native and exotic ant species richness as a function of human population size/density, controlling for country area, plant species richness (as a proxy for habitat heterogeneity), and mean annual temperature and precipitation (variables related to energy availability). Results Ant species richness increased significantly with increasing human population. This result was confirmed when controlling for variations in country area. Both for human population size/density and for ant species richness, there were positive correlations with temperature but not with precipitation. This finding is in agreement with the energy‐availability hypothesis. However, we observed a negative latitudinal gradient in ant and plant species richness, although not in human population size/density. Plant species richness was positively correlated with ant species richness but not with human population size/density. Thus, there is evidence that this type of habitat heterogeneity can play a role in the observed latitudinal gradient of ant species richness, but not in the positive correlation between ant species richness and human population. The results were confirmed for the 545 native and the 32 exotic ant species reported, and we observed a good correlation between exotic and native ant species richness. Main conclusions Ant species richness in European countries conforms to six macroecological patterns: (1) a negative latitudinal gradient; and a positive (2) species–energy relationship, (3) species–area relationship, (4) correlation with plant species richness, (5) exotic–native species richness correlation, and (6) species–people correlation. There is some evidence for the energy‐availability hypothesis, but little evidence for habitat heterogeneity as an explanation of the large‐scale human population–ant biodiversity correlation. This correlation has implications for the conservation of ant diversity in Europe.     

Geographic genetic architecture of pocket gopher (Thomomys bottae) populations in Baja California, Mexico

Phylogenetic analyses of complete mitochondrial cytochrome b sequences support the monophyly of pocket gopher (Thomomys bottae) populations from the 1000 km length of the Baja California peninsula of Mexico, relative to other geographical segments of the species range in western North America. The Baja California peninsula is an area that encompasses considerable ecomorphological and infraspecific diversity within this pocket gopher species. However, detailed population analyses encompassing 35 localities distributed over the southern half of the peninsula reveal only trivial phylogeographical structure. Rather, most of the 72 unique 500-base pair haplotypes examined from 142 individuals is restricted to single populations, although a few haplotypes are shared broadly across geography. Individual populations are typically comprised of haplotype sets from different branches in a network of relationships. Analysis of molecular variance (amova) indicates that approximately half of the total pool of variation is contained among individuals within local populations, and that only about 25% can be explained by the regional subdivisions of current subspecies distributions or physiographic realms. A hypothesized historical vicariant event that has been causally linked to the phylogeographical structure of other, codistributed species has had little influence on these pocket gopher populations, explaining only 13% of the total variation. The temporal depth, estimated by coalescence parameters, of the haplotype lineage in Baja California is relatively recent, approximately 300,000 generations; both the mismatch distribution of pairwise comparisons and a significantly positive exponential growth estimate support a recent history of expanding populations; but current, or recent past, migration estimates have remained small, are largely unidirectional from north to south, and weak isolation by distance is present. All data suggest that pocket gophers have relatively recently invaded the southern half of peninsular Baja California, with the genetic signature of expansion still evident but with sufficient time having lapsed to result in a weak isolation by distance pattern. The geographical assemblage of sampled populations thus appears as a meta-population, with limited gene flow contrasting with random haplotype loss due to drift in small, localized populations.     

Red herring or low illumination? The peninsula effect revisited

Aim The peninsula effect is the prediction that the number of species declines from a peninsula's base to its tip. We evaluated evidence for and against the peninsula effect, and conducted a field study designed to test alternative hypotheses for that effect. Location The Florida peninsula, USA. Methods First, we critically reviewed the accumulated literature on peninsula effects; second, we sampled microcrustaceans in palustrine wetlands on the ridges of peninsular Florida. Site selection in our field study accounted for historical effects and partially controlled for habitat effects. Statistical analyses further accounted for habitat effects, leaving peninsular geometry as the remaining causative mechanism for residual variation in species richness regression analyses. Results Our literature review found mixed evidence (49% of cases) for a peninsula effect. However, most study designs did not control for alternative hypotheses, most comparisons of alternative hypotheses were qualitative, and most studies focused on vertebrate animals. Our field study found that freshwater microcrustaceans inhabiting isolated wetlands on Florida’s peninsular ridges do not exhibit a peninsula effect. Essentially, no variation in microcrustacean species richness could be attributed to peninsular geometry, but 82.5% of variation in species richness was attributed to habitat and sampling effort. Main conclusions Although our research results support the ‘red herring’ label for the peninsula effect, our literature review leads us to argue that more illumination (in the form of study design and quantitative analysis) is needed if mechanisms causing the peninsula effect hypothesis are to be resolved. Future studies of peninsula effects need to control for alternative causative hypotheses (geometry, habitat or history) in study design, and compare quantitatively the effects of hypothesized mechanisms on peninsular diversity patterns. Additionally, studies of taxa other than vertebrate animals need to be conducted for generality. Our study may serve as an example of such an approach.     

Cryptic vicariance in Gulf of California fishes parallels vicariant patterns found in Baja California mammals and reptiles

Comparisons across multiple taxa can often clarify the histories of biogeographic regions. In particular, historic barriers to movement should have affected multiple species and, thus, result in a pattern of concordant intraspecific genetic divisions among species. A striking example of such comparative phylogeography is the recent observation that populations of many small mammals and reptiles living on the Baja California peninsula have a large genetic break between northern and southern peninsular populations. In the present study, I demonstrate that five species of near-shore fishes living on the Baja coastline of the Gulf of California share this genetic pattern. The simplest explanation for this concordant genetic division within both terrestrial and marine vertebrates is that the Baja Peninsula was fragmented by a Plio-Pleistocene marine seaway and that this seaway posed a substantial barrier to movement for near-shore fishes. For some fish species, the signal of this vicariance in mtDNA has been eroded by gene flow and is not evident with classic, equilibrium measures of population structure. Yet, significant divisions are apparent in coalescent analyses that jointly estimate divergence with gene flow. The genetic divisions within Gulf of California fishes also coincide with recognized biogeographic regions based on fish community composition and several environmental factors. It is likely that adaptation to regional environments and present-day oceanographic circulation limit gene exchange between biogeographic regions and help maintain evidence of past vicariance.     

A peninsula as an island: multiple forms of evidence for overwater colonization of Baja California by the gartersnake Thamnophis validus

The recent shift toward dispersal rather than vicariant explanations of disjunct distributions has been driven by the use of molecular data to estimate divergence dates between lineages. However, other kinds of evidence can also be critical in evaluating such biogeographic hypotheses. In the present study, we used a multifaceted approach employing diverse analyses of mitochondrial DNA sequences to assess explanations for the disjunct distribution of the gartersnake Thamnophis validus. The occurrence of this species in the Cape Region of the Baja California peninsula, isolated from mainland populations that occur along the west coast of Mexico, might be explained by: (1) separation of the peninsula from mainland Mexico through rifting 4–8 Mya (tectonic vicariance); (2) fragmentation of the range of this semi‐aquatic species because of post‐Pleistocene aridification (vicariance by aridification); (3) natural overwater dispersal across the Gulf of California; or (4) human introduction. Divergence dating indicates that peninsular and mainland T. validus separated from each other within the last 0.5 Myr, thus rejecting tectonic vicariance. In addition, the estimated closest mainland relatives of peninsular snakes are found farther north than expected under this hypothesis. Three findings argue against vicariance by aridification: (1) peninsular snakes and their closest mainland relatives are more genetically similar than predicted; (2) the location of closest mainland relatives is farther south than predicted; and (3) the species is absent from areas where one might expect to find relict populations. Taken together, refutation of the vicariance hypotheses and the fact that the estimated closest mainland relatives are found almost directly across the Gulf from the Cape Region supports some form of overwater colonization. Various additional arguments suggest that natural dispersal is more likely than human introduction. The present study emphasizes the need for multiple kinds of evidence, beyond divergence dates, to discriminate among hypotheses and to provide independent sources of corroboration or refutation in historical biogeography. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 409–424.