A 12,000 kyr paleohydroclimate record in the southeastern, U.S.A based on deuterium from bat guano

The southeastern United States endures environmental change from human population increase, climate change, and land-use alterations creating the need to understand baseline conditions and environmental patterns prior to human impacts. While paleoenvironmental data can be reconstructed from a variety of archives (e.g., lake sediments, tree rings, speleothems), some geographic areas contain fewer such records. One archive capable of recording moisture regimes and other paleoenvironmental changes over millennia but has received little attention relative to other climate proxies is bat guano deposits in cave systems. Bat guano deposits are found in many cave environments in the southeastern United States and can be used as an archive of paleoenvironmental data including precipitation, vegetation, and aspects associated with the ecology of bats. Here, we present a 12,000-year record of paleoenvironmental change based on δ2H stable isotopes in a guano core collected from Cave Springs Cave in Alabama, USA. Results suggest distinct shifts in moisture with dryer conditions during the early Holocene/late Pleistocene (12,200–9500 cal year BP) (δ2H values − 86.82 to −-77.70), wetter conditions during the middle Holocene (9300–3900 cal year BP) (δ2H values − 125.74 to − 80.63), roughly coinciding with the Holocene Climatic Optimum event time interval (9000–5000 cal year BP). During the last 4000 years, conditions in the region shifted in the southeastern United States region becoming dryer once again. Climate inferences based on guano δ2H are consistent with the role of atmospheric moisture on regional vegetation changes suggested by previous pollen records obtained from lake sediment cores. This study suggests bat guano δ2H may be a reliable method to provide a long-term paleoclimate record.


Introduction
In recent decades, interannual variability in summer precipitation has intensified significantly over the southeastern United States, with an increased frequency of both abnormally wet and dry summers (Wang et al. 2010) primarily due to anthropogenic causes. Summer rainfall is essential to support agriculture, energy production, and the economy of this region (e.g., Manuel 2008;Martinez et al. 2009;Gotvald and McCallum 2010). It is, therefore, crucial to understand the driver of summer precipitation variability from interannual to multidecadal timescales. Regional and global climate models suggest that increasing greenhouse gases can influence summer rainfall patterns in the southeast although the extent to which natural processes versus anthropogenic drivers control rainfall variability remains uncertain (Li et al. 2013).
Paleoclimate and paleoenvironmental records offer the opportunity to investigate the role of natural climate processes on precipitation patterns in the southeastern United States and examine the magnitude of past changes in precipitation and moisture availability. Paleoclimate reconstructions from tree rings, pollen, and lake sediment cores have focused on the polar, temperate, and tropical regions of the globe with fewer records from subtropical areas. The 1 3 148 Page 2 of 13 subtropical southeastern United States is home to environments typically useful for paleoreconstructions (e.g., natural lakes, tree rings), but most records contain short chronologies beginning in the mid-Holocene or later (Larson and Schaetzl 2001). Speleothems provide long chronologies of hydroclimate records using δ 18 O; however, no published speleothem records exist in the southeastern United States. Natural lakes in the southeast are uncommon outside of the coastal plain compared to areas in higher latitudes with histories of glaciers and ice sheets. Most paleoclimate data in the southeastern region have resulted from dendrochronology records (Stahle et al. 1998(Stahle et al. , 2019 which are limited to the most recent millennia (Therrell et al. 2006) or from coastal-plain lakes (Watts 1971(Watts , 1975Whitehead and Sheehan 1985). Current instrumental data of air surface temperatures and rainfall amount, in addition to tree ring chronologies, provide accurate and high-resolution environmental information but do not extend beyond the time interval when the region experienced the first European settlements ~ the 1600-1700s (Stahle et al. 1998;Cleaveland et al. 2003;Therrell et al. 2006).
One documented paleoclimate event that has been supported by pollen records from the southeastern United States is the Holocene Climatic Optimum (HCO) (Delcourt 1980;Delcourt and Delcourt 1980;Folland et al. 1990;Marcott et al. 2013;Tanner et al. 2015;Mendieta et al. 2018). The HCO is characterized by warm periods from 9000 to 5000 years before present (cal year BP) that coincided with a transition from Quercus spp. (oak) dominance to increased Pinus spp. (pine) densities across the southeastern United States landscape as indicated by lake sediment pollen records (Watts 1971;Delcourt 1980;Grimm and Jacobson 1992;Grimm et al. 1993;Watts and Hansen 1994). Additional lake sediment studies have assessed pollen species and abundances to document environmental shifts in vegetation, moisture, and temperature changes over time in the southeast region (Frey 1953;Watts 1971Watts , 1975Filley et al. 2001;Donar et al. 2009). However, few of these studies examined direct moisture regimes from δ 18 O or δ 2 H, which has been used in tropical systems (Curtis et al. 1998;Hodell et al. 1999).
Past climate changes can be reconstructed from lacustrine sediments, ice cores, speleothems, and soils (Grimm et al. 1993;Waters et al. 2009;Medina-Elizalde and Rohling 2012). Isotope time-series from bat guano is one relatively new paleoclimate record that effectively tracks moisture regimes but has received less investigation in the southeastern United States Wurster et al. 2007Wurster et al. , 2010Wurster et al. , 2017Onac et al. 2014;Forray et al. 2015;Cleary et al. 2017Cleary et al. , 2018Cleary et al. , 2019. Bat guano cores can be collected from large, undisturbed guano deposits in cave environments and can be a useful archive for paleoclimate reconstructions Wurster et al. 2007;Onac et al. 2014;Forray et al. 2015;Cleary et al. 2016Cleary et al. , 2018Cleary et al. , 2019Campbell et al. 2017). Guano cores are suitable for study from caves with constant or seasonal bat presence and ideally from areas within the caves that have remained largely isolated from surficial weathering processes (e.g., low ventilation) and human disturbances (Onac et al. 2014;Cleary et al. 2016). In the southeastern United States, unfortunately, these types of deposits are rare since guano mining to produce gunpowder, particularly during the Civil War, depleted the largest/oldest deposits (Hubbard 2019). Many bat species in the southeastern United States migrate seasonally to caves elsewhere, making guano deposits within a single cave seasonally specific (Wurster et al. 2008;Cleary et al. 2016). Previous studies have shown that guano cores record past environmental conditions from an array of climates including tropical, semi-arid, and temperate (Wurster et al. 2017;Campbell et al. 2017;Cleary et al. 2019). When present, guano deposits can provide largely undisturbed depositional environments that can be radiocarbon dated (Onac et al. 2014;Wurster et al. 2017). Primary analyses that have been applied to guano deposits include stable isotopes (Wurster et al. 2008(Wurster et al. , 2010, organic matter (Shahack-Gross et al. 2004;Campbell et al. 2017), pollen (Maher 2006;Geantă et al. 2012;Campbell et al. 2017), elements, such as magnesium, calcium, phosphorous, iron (Giurgiu and Tămas 2013), and nutrients (Hill et al. 1997;Giurgiu and Tămaş 2013).
Bat guano research has been primarily conducted in the southwestern United States (Wurster et al. 2008(Wurster et al. , 2010, the Caribbean (Mizutani et al. 1992a, b;McFarlane et al. 2002;Gallant et al. 2020), Romania (Giurgiu and Tămaş 2013;Onac et al. 2014;Forray et al. 2015;Cleary et al. 2016Cleary et al. , 2018Cleary et al. , 2019, and southeast Asia ). Therefore, the southeastern region of the United States is lacking in guano studies, despite having a large density of caves (over 3000 in the Appalachian region and over 2300 in Alabama) (Culver et al. 1999(Culver et al. , 2006 and roosting bat hibernacula. Currently, only two guano core studies have been published in this region (Campbell et al. 2017;Tsalickis et al. 2021). Given the need for paleoreconstructions in the non-coastal southeastern United States, we analyzed δ 2 H values as well as elements, and nutrients from a guano core collected at Cave Springs Cave in northern Alabama ( Fig. 1) to provide one of the longest records of paleoclimate and paleoenvironmental change for the southeastern United States. Here, we expand upon data described in Tsalickis et al. (2021). The guano core spanned the Holocene time interval (last 11,600 cal year BP) and was collected from a maternal colony of gray bats (Myotis grisescens) that roost in this cave during summer months. From this data, we inferred changes in precipitation and climate patterns from δ 2 H (Deuterium) via bulk guano over the Holocene. Three objectives were investigated: (1) to establish a dated record of isotopic chemistry and precipitation patterns from northern Alabama, (2) to compare the Cave Springs Cave guano record to other paleoclimate records in the southeastern United States to examine their regional context, and (3) to determine environmental drivers of isotopic changes in guano for δ 2 H over time.

Study site and core collection
Cave Springs Cave is located in northern Alabama near the city of Priceville (34.5251° N, 86.8947° W) and is part of the Wheeler National Wildlife Refuge (Fig. 1). Cave Springs Cave is 3371 m in total length and is well mapped. The dominant bat species occupying the cave is the gray bat (Myotis grisescens Howell). The guano pile was ~ 2 m above the cave floor and was cored approximately 487 m from the entrance. The core was retrieved in two sections of polyvinyl chloride (PVC) pipe hammered into the pile with a mallet totaling around 87 cm compacted and 183 cm uncompacted. To prevent overlap and disturbance between the core sections, the guano pile was removed, while the first section was intact so that the beginning of the second section was undisturbed forming one continuous core. Core sections were returned to the lab, stored in a freezer at 0 °C, and were later sectioned at 1 cm intervals. Based on available climate data from the Priceville meteorological station, the present-day climate of the area can be characterized as humid subtropical, with no seasonality in precipitation and monthly averages of ~ 107 mm of rain for 2018. The annual temperature fluctuates between 10 and 32 °C.

Gravimetric analysis, nutrients, and isotopes
Guano core sections were analyzed gravimetrically for bulk density and organic matter. For bulk density, 5 cm 3 of raw guano from each 1 cm interval was weighed, dried, and processed similarly to lake sediments (Brenner and Binford  Campbell et al. 2017). Dried samples of bulk guano from each 1 cm interval were burned at 550 °C for 3 h and organic matter was reported as percent loss on ignition (LOI). Bulk density is reported as g dry/cm 3 wet.
Total phosphorus (P), sulfur (S), and other nutrients (Ca, Fe, Mg, K, Al, Na, S) were measured from dried sediments using an ARL 3560 ICP-AES following complete acid digestion using standard EPA methods (Waters et al. 2009). Total carbon (TC) and total nitrogen (TN) were measured using a Costech combustion elemental analyzer with an attached auto-sampler. Prior to analysis, samples were acidified for 12 h in HCl vapors to remove inorganic carbon.
For δ 2 H analysis, bulk, freeze-dried guano from each 1 cm interval was weighed (~ 1 mg) and folded in tin sample capsules using a Mettler Toledo XPR2U microbalance and sent to the Cornell University Stable Isotope Laboratory (http:// www. cobsil. com/, Ithaca, New York, USA). Deuterium isotopic measurements were calculated using an isotope ratio mass spectrometer (IRMS) relative to an internal standard and calibrated to the international reference standard of V-SMOW (Vienna Standard Mean Ocean Water). The deuterium isotope value is denoted by the delta notation:

Radiocarbon dating
Approximately one gram of guano was sampled from 16, 25, 36, 51, and 56 cm depths in the core. Depths were chosen visually, where abrupt color changes were found. All samples were dried for 24 h in a drying oven and sent to the Center for Applied Isotope Studies, University of Georgia (Athens, Georgia, USA) or National Ocean Sciences Accelerator Mass Spectrometry (AMS) Facility (Woods Hole, MA, USA) for 14 C analysis. AMS 14 C ages were calibrated using the IntCal13 calibration curve as part of the BACON model package in R which uses Bayesian based modeling statistics (Blaauw and Christen 2011) (Table 1). To calculate ages for depths, where 14 C measurements were unavailable, the core top was marked as the date of collection and the uncalibrated 14 C dates were incorporated into the BACON model package in R.

Statistical analyses
Distinctions between early Holocene/late Pleistocene (12,200-9500 cal year BP), middle (9300-3900 cal year BP), and late (3700-0 cal year BP) Holocene were chosen utilizing the δ 2 H measurements from the guano core. A Kruskal-Wallis non-parametric AOV was used for each isotope system using Statistix 9.0, Analytical Software (Tallahassee, FL, USA). A Principal Component Analysis (PCA) was performed on H 2 isotopic values as well as elemental concentrations (Fe, Al, K, Ca, P, and Mg) to determine the ordination of δ 2 H and elements throughout each Holocene period. PCA was conducted using R statistical software.

Core description and age
The Cave Springs Cave guano core was 87 cm in total length when compacted and the area cored from did not show any visual signs of anthropogenic or biological disturbance (e.g., foot traffic and mining). Based on microscopic analysis, the Cave Springs Cave guano core consisted of chitinous insect pieces, bat hair, and fecal material. The bottom of the core was lighter in color and densely packed with clay-like consistency, while the top of the core had recent guano deposits and was dark brown in color with pellet-like consistency (Fig. 2). Between 87 and 67 cm, guano was not observed. Instead, this section of the core was inferred to be the mineral brushite, due to the lack of organic matter and high percentages of P and Ca which are characteristic of brushite found in other guano cores (Giurgiu and Tămaş 2013;Onac et al. 2015). Given the objectives of the study, the brushite part of the core (87-67 cm) was not included in the paleoreconstructions (Giurgiu and Tămaş 2013). From 66 to 42 cm, the guano showed light tan striations. Starting at 41 cm depth, the guano was consolidated and became lighter in color through 28 cm (Fig. 2). The top portion of the core (27-0 cm) was dark brown in color with pellet-like guano (Fig. 2). The Cave Springs Cave guano core provided a dated record of environmental change from present-day to 10,173 ± 45 cal year BP at 56 cm based on calibrated AMS 14 C dates and a BACON model projected age of ~ 12,000 cal year BP at 66 cm (Fig. 3). The measured AMS C 14 dates along with the top of the core were

Elements and nutrients
Organic matter was measured LOI and generally increased up the core (Fig. 4). Organic matter content was the highest at 12,200 cal year BP (71%) and the lowest 4000 cal year BP (22%). Bulk density decreased up the core from 12,200 to 4600 cal year BP corresponding to depth and compaction of the guano through time. Total carbon (TC) increased up the core, containing minimal values of 1.1% at 12,200 cal year BP and a peak value of 39.4% at 600 cal year BP. Total nitrogen (TN) followed the same pattern of increasing up the core and a peak value of 9.1% at 1800 cal year BP. Calcium and phosphorus both were the highest in concentration at the bottom of the core with values of 90.53 mg/g and 54.24 mg/g, respectively. Calcium was most concentrated at 7500 cal year BP as well, while Phosphorus peaks at ~ 12,000 cal year BP (Fig. 4).

δ 2 H isotopes
Between 12,200 and 11,100 cal year BP, the δ 2 H values were consistently less negative averaging − 79.9 ‰, whereas between 10,900 cal year BP and 4200 cal year BP, δ 2 H values were more negative averaging -116.3‰ (Fig. 5). From 4200 cal year BP to the present, δ 2 H values are again less negative (− 77.69 ‰). The transition from the middle to the late Holocene is marked as a pronounced positive shift in δ 2 H.
There were significant differences between early, middle, and late Holocene time periods for δ 2 H (χ 2 = 38.8, p = 0.001). Early Holocene/late Pleistocene and late Holocene were not statistically different based on δ 2 H, whereas middle Holocene was significantly different compared to the early Holocene/late Pleistocene and late Holocene based on δ 2 H. The PCA confirmed that there were three distinct time periods (early Holocene/late Pleistocene, middle, and late Holocene) ordinated differently from each other with 77.5% of the data set's variance accounted for by PC1 and PC2 (Fig. 6).

Dating record
Based on the BACON dating calculations, the Cave Springs Cave guano core spanned the last ~ 12,000 cal year BP and constitutes the oldest guano core environmental record from the southeastern United States. It is important to address only five radiocarbon dates were used to construct the model; however, other studies reconstructing paleoclimate utilizing bat guano have used three or fewer radiocarbon dates Wurster et al. 2010;Onac et al. 2014;Cleary et al. 2016). The comparison of radiocarbon dates between guano core studies has been described in Tsalickis et al. (2021). The calibrated radiocarbon ages contain multiple paleoclimatic events in this region including the HCO. Unlike other sources of paleo data (e.g., lake sediment cores, ice cores) guano cores contain few disturbances related to climate, bioturbation, or redox alteration (Onac et al. 2014;Cleary et al. 2016). The cavern where the guano pile occurred did not show signs of historic flooding or water disturbance as noted in other areas of the cave. As a result, the bat colonies are assumed to have maintained a consistent migratory pattern with summer roosting and did not interact with the guano piles below the roosting areas.

Elemental and nutrient changes
Calcium has the highest concentrations (> 80 mg/g) in the middle Holocene to late Holocene, while Phosphorus has higher concentrations (> 40 mg/g) in the early Holocene/ late Pleistocene. Bird et al. (2007) also documented an increase in phosphorus concentrations in the older portion of his bat guano core, calcium, however, was not measured. Phosphate minerals are common in caves containing bat guano and form due to reactions between guano and clay minerals in acidic conditions (Giurgiu and Tămaş 2013). This could explain why phosphorus and calcium concentrations are highest in the oldest portion of the Cave Springs Cave core-more time allows for phosphate minerals such as brushite, hydroxylapatite, or variscite to form through digestion, dissolution, double replacement, and redox reactions (Onac and Forti 2011;Giurgiu and Tămaş 2013). Therefore, the bottom portion of the Cave Springs Cave core may not be bat guano, but instead cave minerals such as brushite as it is thought that brushite forms from a reaction with guano, calcite, and clay at low pH values (Anthony et al. 2000). However, there are few studies that have examined minerals from bat guano deposits in caves (Fiore and Laviano 1991;Dumitraş et al. 2002Dumitraş et al. , 2004Onac et al. 2004;Marincea et al. 2004;Frost and Palmer 2011).
Following 11,000 YBP, organic matter, carbon, and nitrogen began to decline compared to the rest of the core. This shows a decrease in the mass accumulation rate which could indicate a line of evidence for guano degradation over time and perhaps where diagenesis is beginning to occur in the core. The TC/TN content remains relatively stable throughout the core which may indicate the core is primarily composed of bat guano aside from 12,000 YBP where possible degradation occurs. The carbon, nitrogen, and bulk density also lead us to believe the core is primarily composed of guano and a lack of any sudden changes or shifts in these values indicate bats have been present and using Cave Springs Cave throughout the Holocene without any hiatuses. Although we measured other elements e.g., (Al, Fe, K) and some changes were detected throughout the core, no consistent shifts or patterns were observed, thus, we are confident these elements do not provide any paleoclimatic information. We believe the gray bat population was the dominant bat species using the cave within the past 100 years based on surveys performed by the U.S. Fish and Wildlife Service. While the gray bat is the most common bat today, other insectivorous bats cold have also been encountered in the past. All bat species inhabiting Cave Springs Cave have been insectivorous; therefore, the guano content is consistent throughout the core.

Deuterium evidence in guano studies
Using the δ 2 H values from Cave Springs Cave and based on our statistical analysis, we are distinguishing between wetter versus dryer periods while indicating that there are three distinct shifts in moisture for the region: (1) a dryer period during the early Holocene/late Pleistocene (12,200-9500 cal year BP), (2) a wetter period during the middle Holocene (9300-3900 cal year BP), and (3) a dryer period during the late Holocene (3700-0 cal year BP).
Various factors have been invoked to influence summer precipitation variability in the southeastern United States, including sea surface temperatures (SSTs) and associated atmospheric circulation anomalies (Wang and Enfield 2001;Seager et al. 2003;Schubert et al. 2009;Kushnir et al. 2010), hurricane activity (Elsner and Tsonis 1993;Chan and Misra 2010), soil moisture (Koster et al. 2004;Wu et al. 2007;Wei et al. 2016) and the subtropical high over the North Atlantic (Katz et al. 2003). On interannual to decadal time scales changes in the intensity and position of the North Atlantic subtropical high (NASH) have been linked to precipitation and intensity of drought in the southeastern United States (Li and Li 2011). Studies have recently suggested that the position and intensity of the Fig. 6 Principal Component Analysis of isotope systems (δ 2 H) and elemental concentrations (Fe, Al, K, Ca, P, Mg) in the Cave Springs Cave guano core. D represents δ 2 H. Three distinct time periods are revealed (late Holocene, middle Holocene, and early Holocene/ late Pleistocene) NASH and moisture flux convergence are major drivers of precipitation variability in the southeastern region (Li and Li 2011;Li et al. 2013;Wei et al. 2016). In addition, the southeastern United States was not a known monsoon region during the Holocene and late Pleistocene; therefore, we can assume that meteoric water in this location has remained consistent over time and primarily from precipitation. Thus, we can infer the δ 2 H values here represent the changing hydroclimate record.
Hydrogen (H) isotopes can undergo fractionation through evaporation, condensation, and precipitation as well as through trophic interactions (Schimmelmann and DeNiro 1986;Gröcke et al. 2006;Wurster et al. 2008Wurster et al. , 2010Peters et al. 2012). According to Peters et al. (2012), evaporation favors more negative isotopes; therefore, the water in the bodies of animals, and in this case bats, tends to be deuterium enriched. This causes trophic enrichment of H 2 /H 1 as the H incorporated into animal tissues is from an enriched source. When considering this mechanism in insectivorous bat guano, there is a three-tier or four-tier trophic system possible (plant tissue, insect chitin, bat guano) or (plant tissue, plant-eating insect, carnivorous insect, bat guano) allowing H 2 /H 1 to become enriched by the time it is deposited in bat guano (Gröcke et al. 2006;Onac et al. 2014;Cleary et al. 2016). H 2 /H 1 ratios have frequently been used as measures of paleoclimate (Birchall et al. 2005) despite a lack of understanding trophic discrimination of H isotopes between resources and consumer tissues (Peters et al. 2012). Many paleoclimate studies utilizing bat guano have also not incorporated H 2 /H 1 isotope fractionation through trophic levels into their interpretation Onac et al. 2014;Forray et al. 2015;Choa et al. 2016;Cleary et al. 2016). Wurster et al. (2010) is the first guano study that has applied effects of H 2 /H 1 isotopic fractionation in their guano cores via labile hydrogen exchange with atmospheric H 2 O and reported H 2 /H 1 values from their study fall between − 188 and − 143‰.
Hydrogen isotopic fractionation is a process that was not considered in this study and is also not often considered in other bat guano studies reconstructing paleoclimate Onac et al. 2014;Forray et al. 2015;Choa et al. 2016;Cleary et al. 2016). One reason δ 2 H is not often considered in bat guano studies is due to its high vulnerability to error. δ 2 H profiles can be highly influenced by degradation or contamination, or environmental effects (Wurster et al. 2010). It is accepted that bat guano δ 2 H reflects shifts in moisture source or precipitation amount (Wurster et al. 2008(Wurster et al. , 2010. According to the interpretation of Wurster et al. (2010), more negative δ 2 H values reflect higher precipitation, while less negative δ 2 H values indicate more aridity. The δ 2 H values discussed here are inferred to be driven by moisture rather than temperature.

δ 2 H and pollen comparisons
Pollen records from locations in northwest Georgia, Alabama, Florida, South Carolina, and North Carolina (Frey 1953;Watts 1971Watts , 1975Delcourt 1980) provide evidence for a HCO event in the southeastern United States occurring between 9000 and 5000 cal year BP (Delcourt 1980;Delcourt and Delcourt 1980;Whitehead and Sheehan 1985). The HCO event is coeval with persisting negative guano δ 2 H values interpreted to reflect a wetter period. Pollen records also provide evidence for the HCO which has been frequently observed in previous cores collected from lake sediments across the Gulf Coastal Plain (Frey 1953;Watts 1970Watts , 1971Watts , 1975Delcourt 1979;Delcourt and Delcourt 1980;Whitehead and Sheehan 1985;Watts and Hansen 1994). In addition, Quercus is at its maximum and Pinus at its minimum from 9000 to 5000 cal year BP and this was apparent in lake sediment cores from Bartow County, Georgia, USA (241 km east of Priceville, Alabama) when pine began to dominate about 5000 cal year BP and replaced oak forests during the late Holocene (Fig. 7). Thus, the Quercus to Pinus shift would also be expected to have occurred in northern Alabama with the most likely explanation for this shift to be a change in precipitation. Pinus is better adapted to drier environments (Watts 1971(Watts , 1975Delcourt 1980;LaMoreaux et al. 2009), allowing them to thrive for the last 4000 years, since this is when an inferred decrease in moisture in the Cave Springs Cave δ 2 H record is observed. Quercus dominated from 10,000 to 4000 cal year BP due to wetter conditions (Delcourt 1980;Watts and Hansen 1994), which is also supported by the Cave Springs Cave δ 2 H record.
It is important to take into consideration that pollen may yield different results than bat guano. Pollen producing vegetation can respond to additional environmental factors, such as temperature, topography, soil type, and reproductive patterns. δ 2 H stable isotopes provide a direct measure of moisture, which could cause stratigraphic differences from pollen alterations due to temperature. Furthermore, regional climate differences could cause variations in paleoclimate signals as has been shown from the studies previously mentioned as well as new charcoal records from Florida (Mendieta et al. 2018).
Paleoenvironmental studies based on stable isotope records from wetland and sediment cores disagree on the dominant climate regimes in the southeastern United States across the Holocene. Lake sediment cores from east Georgia, Florida, and the North Carolina and Georgia coastal plains provide evidence for a wetter climate during the early Holocene/late Pleistocene to middle Holocene (Leigh 2008;Filley et al. 2001;Goman and Leigh 2004;LaMoreaux et al. 2009), while pollen records also support a wetter climate during the late to middle Holocene time period (Watts and Hanson 1994;Goman and Leigh 2004;LaMoreaux et al. 2009). Whitehead and Sheehan (1985) describe the middle to early Holocene as being a dryer period and attribute the cause to changes in lightning induced fire frequency and anthropogenic changes in land use in southern Florida based on lacustrine sediments. The δ 2 H guano record reported here is inferred as being linked to precipitation alone, thus differences between pollen and δ 2 H stratigraphies are not surprising given the lack of additional environmental factors recorded in guano. An accepted consensus has not been achieved and additional records are needed to provide a regional pattern.

Conclusions
The bat guano core from Cave Springs Cave in northern Alabama provided a well-dated record of paleoclimate and paleoenvironmental change throughout the Holocene for the summer roosting bat colony. Radiocarbon data revealed this core to be the oldest in the region, providing the longest guano record for the southeastern United States. The most reliable proxy from the Cave Springs Cave core was δ 2 H stable isotopes. Although hydrogen isotope fractionation was not applied to this study (as in other studies), we believe our data supports shifts that corresponded to moisture changes during the late, middle, and early Holocene. In addition, the HCO was determined to be a wetter period, with the deuterium record following closely to the nearby pollen record of Bartow County, Georgia (Watts 1970). Growing research has proven bat guano to be an effective tool for paleoenvironmental reconstructions as it provides a robust and complex resource for interpreting past local climates. In areas of the United States that contain caves with roosting bat colonies (e.g., southeastern United States), bat guano can be an important and promising proxy to use for paleoclimate reconstruction of the local area as well as the broader region.

Fig. 7
Quercus and Pinus pollen record from small ponds in Bartow County, Georgia (Watts 1970) compared to the deuterium record from Cave Springs Cave. The units of measurement for Quercus and Pinus are number of individual specimens (NISP). The gray shaded region indicates the Holocene Climatic Optimum (9000-5000 cal year BP)