Diversity and population characteristics of terrestrial isopods ( Crustacea , Oniscidea ) across three forest environments in southern Brazil

Terrestrial isopods are important and dominant component of meso and macrodecomposer soil communities. The present study investigates the diversity and species composition of terrestrial isopods on three forests on the Serra Geral of the state of Rio Grande do Sul, Brazil. The area has two natural formations (Primary Woodland and Secondary Woodland) and one plantation of introduced Pinus. The pitfall traps operated from March 2001 to May 2002, with two summer periods and one winter. There were 14 sampling dates overall. Of the five species found: Alboscia silveirensis Araujo, 1999, Atlantoscia floridana (van Name, 1940), Benthana araucariana Araujo & Lopes, 2003 (Philoscidae), Balloniscus glaber Araujo & Zardo, 1995 (Balloniscidae) and Styloniscus otakensis (Chilton, 1901) (Styloniscidae); only A. floridana is abundant on all environments and B. glaber is nearly exclusive for the native forests. The obtained data made it possible to infer about population characteristics of this species. The Similarity Analysis showed a quantitative difference among the Secondary forest and Pinus plantation, but not a qualitative one. The operational sex ratio (OSR) analysis for A. floridana does not reveal significant differences in male and female proportions among environments. The reproductive period identified in the present study for A. floridana was from spring to autumn in the primary forest and Pinus plantation and during all year for the secondary forest. The OSR analysis for B. glaber reveals no significant differences in abundance between males and females for secondary forest, but the primary forest was a significant difference. The reproductive period for B. glaber extended from summer to autumn (for primary and secondary forest). This is the first record for Brazil of an established terrestrial isopod population in a Pinus sp. plantation area, evidenced by the presence of young, adults and ovigerous females, balanced sex ratio, expected fecundity and reproduction pattern, as compared to populations from native vegetation areas.

Terrestrial isopods are known as pillbugs and are included in the Oniscidea suborder, presently including 3,939 known species as of 2011 (SchmalfuSS, 2003;Ahyong et al., 2011).The terrestrial isopod fauna in forest ecosystems is important for organic matter processing and nutrient cycling.These organisms are responsible for fragmenting the accumulated leaf litter, along with other members of the detritivore guild (QuadroS & araujo, 2008;Boelter et al., 2009).Many species of terrestrial isopods are not limited to natural environments, with most introduced species associated to anthropogenic environments.Since many species of terrestrial isopods are being transported by humans, they are increasingly being considered synanthropic.
Brazil concentrates a large part of the biodiversity of the planet; however there are many threats to this diversity in various forms of landscape occupation (taBarelli et al., 2005;overBeck et al., 2007).If on one hand monoculture greatly simplifies ecosystems making it hard for local species to establish themselves in these disturbed environments, on the other hand environmental changes due to exotic plant introduction, for example Pinus species, might on its turn favour the establishment of exotic species of terrestrial isopods (magrini et al., 2010) or even native species (Quine & humphrey, 2010).Pinus have been planted in commercial scale in Brazil for over 30 years.Initially, the more extensive areas with plantations occurred in the southern and south-eastern regions, with Pinus taeda and Pinus elliottii.
Tree monocultures, although important economically, result in a series of negative effects to the fauna given the low diversity of resources present (vallejo et al., 1987).The arthropod fauna richness exhibits a tendency to follow plant richness, since many animals depend directly and/or indirectly on plants for survival (Battirola et al., 2007).Areas with ecological dominance from a single tree species can thus be expected to harbour fewer animals and reduced arthropod species diversity (Battirola et al., 2007).This reduction has consequences for ecosystem functioning, as smaller populations of fewer species in the soil fauna would lead to slower and less efficient soil nutrient cycling.
Terrestrial isopods as members of the soil fauna are expected to respond to such environmental changes, although given their limited dispersal abilities, they are probably very sensitive to changes in soil chemical and physical proprieties (paoletti & haSSall, 1999) thus responding rapidly to environmental changes (nakamura et al., 2003) and to a monoculture ageing process (purchart et al., 2013).However, there are no studies on the consequences of Pinus plantation monocultures to populations of terrestrial isopods in Brazil.It is thus important to try and understand not only how many and which species are present in these areas, but also how they are managing to survive in these environments.The impact of Pinus must be evaluated not only in terms of species diversity alone, but also considering the life history of the species present.
Distribution patterns of terrestrial isopods in systems with and without human influence can reveal the purported negative effects of economic land use, helping direct future forest management (lindenmayer & franklin, 2002).Thus, the present study aims to compare the occurrence and distribution of terrestrial isopods in three different nearby forest environments: primary and secondary forest and an old Pinus plantation, in the Serra Geral of the state of Rio Grande do Sul.In addition to diversity data, population information on the terrestrial isopods present were compared among different forest environments, analysing life history traits as reproductive period, fecundity and operational sex ratio (OSR).

Study area.
The "Centro de Pesquisas e Conservação da Natureza Pró-Mata" (heretofore Pró Mata, between 29°27' -29°35'S and 50°08' -50°15'W), is located in São Francisco de Paula municipality, state of Rio Grande do Sul, southern Brazil.This area is the stage for the contact of three ecoregions (as defined by the WWF, olSon et al., 2001): moist araucaria forest (NT 0101), alto Paraná Atlantic forests (NT 0150) and the Uruguayan savannah (NT 0710, represented by altitude savannah ecotones).Besides the vegetation typical of those ecoregions, there is the recorded presence of some species from the Semidecidual Seasonal Forest, found to the southwest (Bertoletti & teixeira, 1995), and a growing area has been occupied by Pinus silviculture.The soil is typical of the Serra Geral Formation, with a thick sequence of vulcanites, eminently basaltic, containing effusive acid rocks, abundant at the top.Geomorphologically this area is in the east border of the Araucaria highlands (Bertoletti & teixeira, 1995).
Sampling.Three different forest ecosystem types were sampled with simple replication resulting in six areas: primary forest (with little human disturbance -two blocks of approximately 5.5 and 9.5 ha, 2.7 km apart from each other), secondary forest (with some human disturbance in the near past, but not in the last 15 years -two blocks of 4 ha and 4.3 ha, 2.9 km apart from each other) and Pinus plantation (Pinus taeda Linnaeus and Pinus elliottii Engelm -respectively around 3 ha and 10 ha blocks, 400 m apart from each other).The primary forest has the marked presence of Araucaria angustifolia (Bert.)O. Kuntze, a tall, canopy conifer species.The secondary forest is in the highland slope areas of Pró-Mata, with rare large trees and bamboo patches interspersed leaving this latter component as an important part of the leaf litter.The Pinus was planted long ago, in 1950, having thus a shrub layer formed by juvenile and small individuals of native tree species (Souza & lorenzi, 2008).In each of the six areas two subareas were chosen, on which two independent transects were placed at last 50 apart from each other.For each transect there were five pitfall traps 10 m apart from each other.The preserving liquid was formaldehyde 4% with drops of domestic detergent to minimise surface tension.The liquid was renewed every other 30 days.Traps operated from March 2001 to May 2002, with two summer periods and one winter.There were 14 sampling dates overall.Trap contents were brought to the lab and searched throughout to separate the isopods.
Data analysis.Isopod abundance and species richness was compared among environments using a oneway ANOVA.Likewise, an ANOSIM (analysis of similarity, clarke, 1993) with 10,000 permutations was employed to test for differences in species composition among forest environments.Both a quantitative (Bray-Curtis) and qualitative (Simpson) index were used to clarify the influence species abundances has on species composition.To identify the taxon responsible for differences between two or more environments and to quantify its contribution to this difference a SIMPER (Similarity Percentage) analysis was used (clarke & WarWick, 1994).All analyses were implemented with Past software (hammer et al., 2003).
The two most frequent species were investigated regarding their operational sex ratio (OSR) (emlen & oring, 1977), fecundity and reproductive period.To calculate the OSR ovigerous and post-ovigerous females were excluded, since having a marsupium they are no longer eligible for a new copulation (araujo & Bond-Buckup, 2005).Females with cephalothorax length lower than that of the smaller ovigerous female found in the literature (araujo & Bond-Buckup, 2005) were not considered either.A G-test (Sokal & rohlf 1995) was employed to verify differences among forest areas in isopod sex ratio, and against the neutral (Fisherian) expectation of a 1:1 sex ratio.Females were divided in three size classes: small, medium and large, to estimate average fecundity.The reproductive period was identified as the time of the year when ovigerous and post-ovigerous females were present.

RESULTS
Overall there were 622 individuals, belonging to five species of terrestrial isopods: Alboscia silveirensis Araujo, 1999, Atlantoscia floridana (van Name, 1940) and Benthana araucariana Araujo & Lopes, 2003 (Philoscidae), Balloniscus glaber Araujo & Zardo, 1995 (Balloniscidae) and Styloniscus otakensis (Chilton, 1901) (Styloniscidae).These species represent 35.7% of the known Oniscidea fauna for the Serra Geral of state of the Rio Grande do Sul.The ANOVA revealed no differences among the areas either for abundance (F 4.25 = 1.604; p = 0.254) (actually an index of activity) or richness (F 4.25 = 1.016; p = 0.400) (Fig. 1).Atlantoscia floridana was abundant in all areas, with the second most abundant being B. glaber, significantly present in primary and secondary forests.Of the five recorded species, only A. silveirensis was not recorded from the Pinus plantations (Tab.I).Some species showed very low abundance, being represented by few individuals, especially when considering environments separately.The primary forest had A. silveirensis as a doubleton; the secondary forest had three singletons (A.silveirensis, B. araucariana and S. otakensis) and Pinus plantations had one singleton (B.glaber) and one doubleton (B.araucariana).
The ANOSIM revealed a quantitative difference between secondary forest and Pinus plantation (Tab.II), but not a qualitative difference (Tab.III), that is, the species identities do not change much between these environments, but their abundances do.Atlantoscia floridana abundance changes among environments were the main responsible for these quantitative composition differences among environments (SIMPER -Similarity Percentage, Tab.IV).
All classes of size A. floridana are present in all environments.The operational sex ratio (OSR) analysis for A. floridana does not reveal significant differences in male and female proportions among environments (primary forest: χ 2 = 0.097; df = 1; p = 0.756; secondary forest: χ 2 = 3.571; df =1; p = 0.059; Pinus plantation: χ 2 = 1.067; df = 1; p = 0.302).The G-test on the expected Fisherian sexual proportion (1:1) is not significant either (G = 2.168; df = 1; p = 0.141).The smallest ovigerous A. floridana female occurred in the Pinus plantation (0.968 mm), with the largest size found in the secondary forest (1.800 mm).Data on isopod fecundity appears in Table V.The reproductive period identified in the present study for A. floridana was from spring to autumn in the primary forest and Pinus plantation and during all year for the secondary forest.There were no significant differences in the abundance of ovigerous and post-ovigerous females among environments (ANOVA, F 3,23 = 2.042; p = 0.143).
The second most frequent species, Balloniscus glaber, is more abundant in primary and secondary forests, with all age classes found there.In Pinus plantations there was a single female recorded in the summer of 2001.The OSR analysis for B. glaber reveals no significant differences in abundance between males and females for secondary forest (χ 2 = 2.769; df = 1; p = 0.096); for the primary forest, however, there was a significant difference (χ 2 = 11.25;df = 1; p = 0.0008).The G-test again showed differences from the expected 1:1 Fisherian sex ratio (G = 12.645; df = 1; p = 0.0004).For this species the fecundity equation was determined, as well as the minimum and maximum number of eggs/mancas found in ovigerous females according to size classes on each environment (Tab.VI).In this case females were considered ovigerous only when having a completely closed marsupium, and all females with any opening in the marsupium were considered post-ovigerous.The smaller and larger ovigerous female occurred in the primary forest (1.59 mm and 2.5 mm respectively).There were no significant differences regarding ovigerous and post-ovigerous female abundance among environments (ANOVA F 4,22 = 0.2014; p = 0.657).The reproductive period for B. glaber extended from summer to autumn (for primary and secondary forest).

DISCUSSION
This is the first record for Brazil of an established terrestrial isopod population in a Pinus sp.plantation area, evidenced by the presence of young, adults and ovigerous females, balanced sex ratio, expected fecundity and reproduction pattern, as compared to populations from native vegetation areas.It is also the first isopod diversity study to complement such diversity data with information on isopod life history traits.Of the five species found, only A. floridana is abundant on all environments.Balloniscus glaber is nearly exclusive for the native forests, the single individual in the Pinus area probably a "tourist" (dispersing through the area instead of inhabiting it).The other species had low abundances on every environment.Benthana araucariana, a species originally described for this ecoregion is unexpectedly rare on all environments.Styloniscus otakensis is an exotic species, but also not abundant.
Terrestrial isopod diversity is associated to the availability and quality of the food sources present (zimmer, 2003) and also the variety of resources and microhabitats the environment offers (lavelle, 1996; irmler, 2000).However, human disturbance in the studied environments, either through the monoculture of an exotic species or changes induced to native forest, ends up changing the expected environmental structure and function (morriS, 2003).Pinus plantations offer a smaller amount of resources for the fauna, not only generating a low quality leaf litter but also restricting litter shelter (loureiro et al., 2006).A Pinus plantation in a given area does not lead to complete absence of terrestrial isopods, as both species richness and abundance did not differ from the native forests.The presence of a developed understory in this old Pinus area can be actually ameliorating this environment, guaranteeing more microhabitats and better food resources, attracting more isopods, especially habitat generalists.A similar scenario was found in Britain, where well-established plantations of exotic trees were considered emergent ecosystems providing habitat for native species and considered important for biodiversity (Quine & humphrey, 2010).This would explain A. floridana indiscriminate abundance across environments since it is known to have a strong capacity for coping with different, stressful situations (QuadroS et al., 2009).
The quantitative difference in species composition between secondary forest and Pinus plantation would indicate that richness and abundance of soil arthropods are larger in native forests than Pinus monoculture, but the primary forest being similar to Pinus leaves the question open.Actually these two environments have important characteristics in common: the strong predominance of a conifer (A.angustifolia in the primary areas) and the importance of these trees for litter formation are coupled with the above mentioned developed understory in the Pinus areas chosen here.These could perhaps explain the lack of difference in isopod fauna.More recent Pinus plantation areas would probably offer less quantity and lower quality of resources for terrestrial isopods and can thus still be expected to be biologically poorer.
Very rare species in each environment (local singletons and local doubletons) can be thought of as occasional or "vagrant" (lucky et al., 2002), which would mean instead of a local population, an occasional capture revealing dispersal through an inadequate environment.Invertebrate diversity studies in tropical forests indicate a high number of singletons to be common (coddington et al., 2009).Such evidence reinforces the need for considering species life history data when analyzing diversity data.Despite our knowledge on the biology of the rare species found in this study being scant, the low numbers may also reflect a sampling bias.Alboscia silveirensis, for example, is endogeous (QuadroS et al., 2007) being thus found below the superficial soil layer or within rotting logs.These habitat preferences allow us to consider this species as a true occasional in the present study, despite the possibility of it being rare as well.
Environmental preferences of the main species found in this study corroborate the existing information: A. floridana and B. glaber are abundant species in terrestrial isopods assemblages in southern Brazil (lopeS et al., 2005;almerão et al., 2006;QuadroS & araujo, 2008).Even considering distinct sampling methods, these species can be confirmed as common for the state of the Rio Grande do Sul.
Atlantoscia floridana is a generalist and can occupy a gamut of habitats: bromeliads, Polyporaceae fungi, ant nests, rotting logs, litter in forests, including fallen banana tree and coconut tree leaves, underneath stones, coconut skins and decaying matter in general (lemoS de caStro, 1985).Given this high level of adaptation, it is no surprise this species was found in the three environments studied here.Atlantoscia floridana life history traits favour a fast habitat colonisation rate (QuadroS et al., 2008) justifying its presence even in Pinus plantations, with relatively high abundances, as in most cases (almerão et al., 2006).A proper population in a Pinus plantation can be due, as argued above, to a well-developed understory, characteristic of old or abandoned plantation, thus losing the aspect of a monoculture.
Habitat generalist species tend to occupy areas even though food quality is not adequate.Pinus leaf litter has many components impalatable to edaphic animals (vink & purWanti, 1994), which raises the need for increasing litter palatability and digestibility by weakening its physical and chemical structure through microorganismal actions (haSSall & ruShton, 1984).In the Pinus case, fungi can be ingested along with litter (Soma & Saitô, 1983;haSSall & ruShton, 1984).Terrestrial isopods are primarily detritivores, but in the absence of nutritive food, fungi can become a viable option.The high frequency of soil fungi characteristic of monocultures as Pinus and Eucalyptus plantations lends credence to the observed proliferation of fungivores (fonSeca et al., 2009) and also occasional consumers such as terrestrial isopods.
The information on A. floridana reproduction patterns and sex ratio complements what is found in the literature: A. floridana does not have a clear reproductive peak during winter on any environments.The fecundity of this species in the present study was relatively lower (2 to 20 eggs) than the known values of 4 to 23 eggs (araujo & Bond-Buckup, 2005;QuadroS et al., 2008).The smaller ovigerous female of A. floridana found in the Pinus plantation, along with the lower fecundity, is probably associated with low food quality in Pinus (Smith & Bradford, 2003), resulting in a faster and lower reproductive investment.
The biology of B. glaber has been studied in detail in the latter years (almerão et al., 2006;meinhardt et al., 2007;QuadroS & araujo, 2007QuadroS & araujo, , 2008;;QuadroS et al., 2009;QuadroS, 2010).It is known to be a habitat specialist (QuadroS et al., 2008), justifying it's nearly absence in the Pinus plantation.Its geographic distribution is restricted and it has been recorded only from preserved areas or those with little human influence (araujo & zardo, 1995).The absence of different age classes in the Pinus plantation, especially ovigerous females, suggests B. glaber does not find the conditions adequate for establishment there.The fecundity here was higher (4 to 31 eggs) than previously recorded (5 to 20 eggs) (QuadroS et al., 2008).In this region the reproductive period of B. glaber includes autumn, differently from the previous record of reproduction only during spring and summer (QuadroS et al., 2008).
The search for edaphic fauna responses to the negative impact of Pinus plantations must be associated to a wealth of information on the target organisms.Not only species diversity data can be revealing, but life history traits can show in more detail how the biology of certain species is affected.Here we have provided records of how terrestrial isopods are living in these environments, and this can serve as a basis for future diversity studies on this group.Monitoring terrestrial isopods in the soil-leaf litter system of Pinus plantation areas can result in relevant data important for the management and restoration of disturbed areas.